WO2022177104A1 - Method for producing exosomes having enhanced biological activity, and application thereof - Google Patents
Method for producing exosomes having enhanced biological activity, and application thereof Download PDFInfo
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- WO2022177104A1 WO2022177104A1 PCT/KR2021/017394 KR2021017394W WO2022177104A1 WO 2022177104 A1 WO2022177104 A1 WO 2022177104A1 KR 2021017394 W KR2021017394 W KR 2021017394W WO 2022177104 A1 WO2022177104 A1 WO 2022177104A1
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- exosomes
- ascorbic acid
- physiological activity
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present invention relates to a method for producing exosomes, and to a method for producing exosomes with enhanced physiological activity.
- the present invention relates to a method for enhancing the physiological activity of exosomes, and a medium for the same.
- the present invention relates to the application of the exosomes with enhanced physiological activity obtained by the production method as described above.
- cell secretome contains various bioactive factors that regulate cell behavior.
- cell secretion contains 'exosome' or Because it contains 'extracellular vesicle', research on its components and functions is being actively conducted.
- extracellular vesicles Cells release various membrane types of ERs to the extracellular environment, and these ERs are commonly referred to as extracellular vesicles (EVs).
- the extracellular vesicles are also called cell membrane-derived ERs, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, they are used separately from exosomes.
- Exosomes are endoplasmic reticulum with a size of several tens to hundreds of nanometers made of a double phospholipid membrane identical to the structure of the cell membrane, and the inside contains proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo.
- Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells.
- Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells.
- Exosomes contain specific genetic material and bioactive factors according to the nature and state of the cell from which they are derived. In the case of proliferating stem cell-derived exosomes, it regulates cell behaviors such as cell migration, proliferation and differentiation, and reflects the characteristics of stem cells related to tissue regeneration (Nature Review Immunology 2002 (2) 569-579).
- exosomes called avatars of cells contain bioactive factors such as growth factors similar to cells, and serve as a carrier for transporting bioactive factors between cells, that is, communication between cells.
- Exosomes are known not only to be released from animal cells such as stem cells, immune cells, fibroblasts and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae. .
- Another object of the present invention is to provide a method for enhancing the physiological activity of exosomes.
- Another object of the present invention is to provide a medium for enhancing the physiological activity of exosomes.
- Another object of the present invention is to provide various applications of the exosomes with enhanced physiological activity obtained by the production method as described above.
- the present inventors have been repeating intensive studies on a method for producing exosomes with enhanced physiological activity, and when cells are cultured in a medium containing ascorbic acid, an analog or derivative thereof, the physiological activity of the exosomes isolated from the culture medium is improved It was confirmed that the present invention was completed.
- extracellular vesicles generally encompasses membrane vesicles, ectosomes, shedding vesicles, microparticles, or equivalents thereof. used in the sense that Depending on the isolation environment, conditions and methods, etc., the extracellular vesicle may have the same meaning as an exosome, and may have the same or similar size as the exosome, but may also have a meaning including nanovesicles that do not have the composition of the exosome.
- the term exosome used herein is used to encompass the extracellular vesicles.
- exosomes refers to endoplasmic reticulum with a size of several tens to hundreds of nanometers (preferably about 30 to 200 nm) consisting of a double phospholipid membrane identical to the structure of the cell membrane (provided that the separation target is The particle size of exosomes may vary depending on the type of cell to be used, separation method and measurement method) (Vasiliy S. Chernyshev et al., “Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015)) DOI 10.1007/s00216-015-8535-3). Exosomes contain proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo.
- Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells.
- Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known
- exosome used herein has a nano-sized vesicle structure secreted from animal-derived cells and released into the extracellular space and has a composition similar to that of exosomes (eg, exosomes). -It means to include all similar vesicles).
- the type of animal-derived cells from which the exosomes are derived is not limited, but may be stem cells or immune cells as an example not limiting the present invention.
- the stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, embryonic stem cell-derived mesenchymal stem cells, or induced pluripotent stem cell-derived mesenchymal stem cells.
- the immune cells may be T cells, B cells, NK cells, cytotoxic T cells, dendritic cells or macrophages.
- the adult stem cells include one or more adult stem cells selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and pluripotent stem cells. It may be a stem cell.
- the mesenchymal stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton's jelly and placenta.
- the adult stem cells may be mesenchymal stem cells, for example, stem cells derived from fat, bone marrow, umbilical cord or umbilical cord blood, and more preferably, adipose-derived stem cells.
- the type of the stem cell or the immune cell is not limited as long as there is no risk of infection by a pathogen and does not cause immune rejection, but preferably a human stem cell or human-derived immune cell.
- adipose stem cells used in the examples described below should be understood as an example of animal cells that can be used in the present invention, and the present invention is not limited thereto.
- ascorbic acid an analog or derivative thereof
- ascorbic acid ascorbic Acid
- ascorbic acid-2-glucoside Ascorbic acid-2-glucoside
- ascorbic acid-2 -Sulphate Ascorbic acid-2-sulfate
- AA-2S ascorbic acid-2-phosphate
- magnesium ascorbyl phosphate Magnnesium ascorbyl phosphate
- ascorbyl palmitate Ascorbyl palmitate
- Retinyl Ascorbate Retinyl Ascorbate
- Tetrahexyldecyl Ascorbate Tetrahexyldecyl Ascorbate
- Sodium ascorbate Sodium ascorbate
- Calcium ascorbate Calcium ascorbate
- Polyethoxylated ascorbate Polyethoxylated ascorbate (Polyethoxylated ascorbate) acid)
- Ethyl Ascorbic Acid Aminopropyl Ascorbate
- pretreatment refers to culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof
- pretreatment exosome or ascorbic acid-treated exosome refers to ascorbic acid, an analog or derivative thereof. It refers to an exosome obtained by culturing animal-derived cells in a containing medium.
- Untreated means culturing animal-derived cells in a medium that does not contain ascorbic acid, an analog or derivative thereof
- untreated exosomes, untreated control exosomes or untreated control refers to the cell culture step and exo It refers to exosomes obtained without treatment with ascorbic acid, an analog or a derivative thereof, in any step of the isolation step.
- anti-inflammatory means preventing, inhibiting, alleviating, ameliorating or treating inflammation
- inflammatory diseases are examples that are not limited to the present invention, and include dermatitis, atopic dermatitis, eczema, bacterial infection, virus Inflammation due to infection or fungal infection, burns, inflammation due to burns, wounds, inflammation due to wounds, ulcerative colitis, arthritis, rheumatoid arthritis, hepatitis, nephritis, and the like.
- wound refers to a state in which a living body is damaged, and a tissue constituting the internal or external surface of the living body, for example, skin, muscle, nerve tissue, bone, soft tissue, internal organ, or vascular tissue It encompasses segmented or disrupted pathological conditions.
- the wound is abrasion, laceration, cut, cut, avulsion, pressure sore, pit wound, tissue destruction by radiation, penetrated wound, gunshot wound ( gun shot wound), burns, frostbite, surgical wounds, sutures after plastic surgery, chemical wounds, etc., and may include damage to any part of an object.
- Iontophoresis refers to a method of allowing an ionized active ingredient to permeate the skin with an electrical repulsive force by flowing a microcurrent to the skin to which an active material is applied to change the electrical environment of the skin by giving a potential difference means
- Iontophoresis used in one embodiment of the present invention is a method in which a current from an external power source flows into an electrode patch on the skin to introduce a microcurrent to the skin, and a battery is mounted on the electrode patch itself to give the skin
- a method in which microcurrent is introduced, a method in which microcurrent is introduced into the skin through a patch equipped with a reversed electrodialysis means that generates an electric current through a difference in ion concentration between a high-concentration electrolyte solution and a low-concentration electrolyte solution, etc. can
- the present invention is not limited thereto, and it goes without saying that various types of iontophoresis may be used.
- the present invention provides a method for enhancing the physiological activity of exosomes, comprising culturing an animal-derived cell in a medium containing ascorbic acid, an analog or derivative thereof.
- the method for enhancing the physiological activity of an exosome of one embodiment of the present invention may include further culturing the animal-derived cells in a serum-free medium containing ascorbic acid, an analog or a derivative thereof.
- the concentration of the ascorbic acid, an analog or derivative thereof is about 1 ⁇ g/mL to 300 ⁇ g/mL, preferably about 10 ⁇ g/mL to 100 ⁇ g/mL mL.
- the enhancement of the physiological activity may be anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancement.
- the enhancement of the physiological activity may be enhancement of the effect of increasing collagen synthesis, for example, may be at least one of skin elasticity enhancement enhancement, wrinkle improvement enhancement enhancement, or skin regeneration enhancement enhancement.
- the present invention provides a medium for enhancing the physiological activity of exosomes derived from animal cells, comprising ascorbic acid, an analog or derivative thereof.
- the enhancement of the physiological activity may be anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancement.
- the enhancement of the physiological activity may be enhancement of the effect of increasing collagen synthesis, for example, may be at least one of skin elasticity enhancement enhancement, wrinkle improvement enhancement enhancement, or skin regeneration enhancement enhancement.
- the concentration of the ascorbic acid, an analog or derivative thereof is about 1 ⁇ g/mL to 300 ⁇ g/mL, preferably about 10 ⁇ g/mL mL to 100 ⁇ g/mL.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising exosomes with enhanced physiological activity obtained by culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof.
- the exosomes with enhanced physiological activity may be exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy
- the pharmaceutical composition includes the exosomes with enhanced physiological activity as an active ingredient, anti-inflammatory , it may be a pharmaceutical composition for treating wounds or promoting wound healing.
- the pharmaceutical composition of one embodiment of the present invention comprises the steps of (a) culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and (b) secreted or released from the animal-derived cells. It includes exosomes with enhanced anti-inflammatory, wound treatment or wound healing promoting efficacy, obtained by performing the step of separating the exosomes from the collected culture medium after collecting the conditioned media containing the exosomes.
- the ascorbic acid, analog or derivative thereof is present in the medium at a concentration of about 1 ⁇ g/mL to 300 ⁇ g/mL, preferably about 10 ⁇ g/mL to 100 ⁇ g/mL. concentration may be included.
- the pharmaceutical composition of one embodiment of the present invention may be administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof.
- the pharmaceutical composition may be in the form of an injection, injection, spray, liquid or patch.
- the pharmaceutical composition of one embodiment of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent.
- the carrier, excipient and diluent include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto. .
- Administration of the pharmaceutical composition of one embodiment of the present invention means introducing a predetermined substance to the patient by any suitable method, and the administration route of the pharmaceutical composition is administered through any general route as long as the drug can reach the target tissue.
- the pharmaceutical composition of one embodiment of the present invention may be administered orally or parenterally, and parenteral administration includes intra-articular administration, intra-articular administration, intrasynovial administration, intrasternal administration, intrathecal administration.
- parenteral administration includes intra-articular administration, intra-articular administration, intrasynovial administration, intrasternal administration, intrathecal administration.
- administration intralesional and intracranial administration, transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intralymphatic administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, rectal administration, etc.
- the pharmaceutical composition of one embodiment of the present invention can be administered by any device capable of transporting an active substance to a target tissue or cell.
- the effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to expect anti-inflammatory, wound healing, and/or wound healing promoting effects.
- the formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilized formulation, or a suppository.
- the formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may also be prepared as an injection.
- the injection of one embodiment of the present invention may be an aqueous injection, a non-aqueous injection, an aqueous suspension injection, a non-aqueous suspension injection, or a solid injection used by dissolving or suspending, but is not limited thereto.
- the injection of one embodiment of the present invention is distilled water for injection, vegetable oil (for example, peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, benzoate estradiol, bismuth salicylate, depending on the type , Arsenobenzol sodium, or at least one of streptomycin sulfate, and may optionally include a stabilizer or a preservative.
- the blending ratio of the pharmaceutical composition of one embodiment of the present invention can be appropriately selected according to the type, amount, form, etc. of the additional ingredients as described above.
- the pharmaceutical composition of the present invention may be included in about 0.1 to 99% by weight, preferably about 10 to 90% by weight.
- a suitable dosage of the pharmaceutical composition of one embodiment of the present invention may be adjusted depending on the severity of the disease, the type of formulation, the formulation method, the patient's age, sex, weight, health status, diet, excretion rate, administration time and administration method. can For example, when the pharmaceutical composition of one embodiment of the present invention is administered to an adult, it may be administered in one to several divided doses at a dose of 0.001 mg/kg to 100 mg/kg per day.
- the present invention provides a cosmetic composition
- a cosmetic composition comprising exosomes with enhanced collagen synthesis-increasing efficacy obtained by culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof.
- the cosmetic composition may be, for example, a cosmetic composition for skin elasticity improvement, wrinkle improvement and/or skin regeneration.
- the cosmetic composition of one embodiment of the present invention comprises the steps of (a) culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and (b) secreted or released from the animal-derived cells. After collecting the culture medium containing the exosomes (conditioned media), it includes exosomes with enhanced collagen synthesis effect obtained by performing the step of separating the exosomes from the collected culture medium.
- the ascorbic acid, analog or derivative thereof is present in a concentration of about 1 ⁇ g/mL to 300 ⁇ g/mL in the medium, preferably about 10 ⁇ g/mL to 100 ⁇ g/mL. concentration may be included.
- the cosmetic composition may include shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, It may be a mist or powder, and preferably a lotion or cream.
- the cosmetic composition of one embodiment of the present invention is a component commonly used in a cosmetic composition within a range that does not impair the effects of the present invention, for example, a moisturizer, an antioxidant, an oily component, a UV absorber, an emulsifier, a surfactant, a thickener , alcohols, powder components, colorants, water-based components, water, and various skin nutrients can be appropriately blended as needed.
- a moisturizer for example, a moisturizer, an antioxidant, an oily component, a UV absorber, an emulsifier, a surfactant, a thickener , alcohols, powder components, colorants, water-based components, water, and various skin nutrients can be appropriately blended as needed.
- the cosmetic composition of one embodiment of the present invention in addition to exosomes with enhanced physiological activity, conventionally used skin as long as it does not impair its action (eg, skin elasticity improvement, wrinkle improvement and/or skin regeneration, etc.)
- An elasticity improving agent, an anti-wrinkle agent, and/or a moisturizing agent may be mixed and used together.
- the cosmetic composition comprising the exosome with enhanced physiological activity of the present invention as an active ingredient is at least one of hydrogel, hyaluronic acid, hyaluronic acid salt (eg sodium hyaluronate, etc.), or hyaluronic acid gel may be supported or mixed.
- the type of the hydrogel is not limited, but may preferably be a hydrogel obtained by dispersing a gelling polymer in a polyhydric alcohol.
- the gelling polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum.
- the polyhydric alcohol may be at least one selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol and glycerin.
- the cosmetic composition of one embodiment of the present invention is, for example, a patch, mask pack, mask sheet, cream, tonic, ointment, suspension, emulsion, paste, lotion, gel, oil, pack, spray, aerosol, mist, foundation, It can be applied to various forms such as powder and oil paper.
- the cosmetic composition may be applied or deposited on at least one surface of a patch, mask pack, or mask sheet.
- the cosmetic composition of one embodiment of the present invention may be used for the purpose of skin elasticity improvement, wrinkle improvement and/or skin regeneration, and the cosmetic formulation may be prepared in any formulation conventionally prepared in the art.
- the cosmetic composition of one embodiment of the present invention includes components commonly used in the cosmetic composition, for example, it may include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances.
- other components can be appropriately selected and formulated by those skilled in the art without difficulty depending on the type or purpose of use of the cosmetic composition.
- the present invention exhibits an excellent effect capable of enhancing the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes or physiological activities such as collagen synthesis. Therefore, the method for producing exosomes with enhanced physiological activity or the method for enhancing the physiological activity of exosomes of the present invention can produce commercially and/or clinically useful exosomes with enhanced physiological activity, and thus produced physiological activity This reinforced exosome can be usefully utilized as an active ingredient in a pharmaceutical composition or cosmetic composition.
- 1A is a graph showing the particle size distribution and the number of particles obtained by performing NTA (nanoparticle tracking analysis) on exosomes obtained after pretreatment and culturing of stem cells in a medium containing ascorbic acid, an analog or derivative thereof.
- 1B is a graph showing the particle size distribution and the number of particles obtained by performing NTA (nanoparticle tracking analysis) on exosomes obtained after pretreatment and culturing of stem cells in a medium not containing ascorbic acid, analogs or derivatives thereof. .
- Figure 2 is an anti-inflammatory of the exosomes obtained after pretreatment and culturing of stem cells in a medium containing ascorbic acid, an analog or derivative thereof according to an embodiment of the present invention (hereinafter, referred to as "ascorbic acid-treated exosomes”) It is a comparative graph showing that the efficacy is significantly increased compared to the exosomes obtained after culturing the stem cells in a medium not containing ascorbic acid, analogs or derivatives thereof (hereinafter, referred to as "untreated control exosomes").
- FIG. 3 is a comparative graph showing that the collagen synthesis promoting effect of the ascorbic acid-treated exosome of the present invention is significantly increased compared to that of the untreated control exosome.
- FIG. 4 is a comparative graph showing that the cell proliferation promoting effect of the ascorbic acid-treated exosomes of the present invention is significantly increased compared to the untreated control exosomes.
- Human adipose-derived stem cells were suspended in DMEM culture medium containing 10% fetal bovine serum (FBS), 100 Units/mL penicillin, and 100 ⁇ g/mL streptomycin, and then inoculated into a flask. 37° C., 5% CO 2 Incubated in an incubator. In the case of the ascorbic acid-treated group, 30 ⁇ g/mL of ascorbic acid was added to the culture medium.
- FBS fetal bovine serum
- penicillin 100 Units/mL
- streptomycin 100 ⁇ g/mL
- both the ascorbic acid-treated and untreated groups reached a cell confluency of 80% or more, the cells were washed with phosphate-buffered saline (PBS, purchased from Thermo Scientific), and the culture medium was cultured at 100 units/mL.
- PBS phosphate-buffered saline
- SFM serum-free media
- the cells were cultured for 24 hours to 72 hours, and the supernatant (hereinafter, culture solution) was collected, and the cell count was measured using a cell counter for the collected cells.
- culture solution hereinafter, culture solution
- RAW 264.7 a mouse macrophage cell line
- DMEM purchased from Thermo Scientific
- fetal bovine serum purchased from Thermo Scientific
- antibiotics-antimycotics purchased from Thermo Scientific
- human dermal fibroblasts and a medium for culturing them were purchased from Cell Bio Co., Ltd. (Seoul, Korea) and subcultured at 5% CO 2 , 37°C conditions.
- the culture solution recovered in Example 1 was filtered with a 0.22 ⁇ m filter to remove impurities such as cell debris, wastes, and large particles.
- the filtered culture solution was separated from the exosomes through a separation process using tangential flow filtration (TFF).
- TFF tangential flow filtration
- a cartridge filter aka hollow fiber filter; purchased from GE Healthcare or Spectrum Labs
- a cassette filter purchased from Pall or Sartorius or Merck Millipore
- TFF filters can be selected by various molecular weight cutoff (MWCO). Exosomes were selectively separated and concentrated by the selected MWCO, and particles smaller than the MWCO, proteins, lipids, nucleic acids, and low molecular weight compounds were removed.
- a TFF filter of MWCO 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da, or a TFF filter having a size of 0.05 ⁇ m was used. While the culture medium was concentrated to a volume of about 1/100 to 1/10 using the TFF method, substances smaller than MWCO were removed to separate the exosomes.
- the separated and concentrated exosome solution was further desalted and buffer exchanged (diafiltration) using the TFF method.
- desalting and buffer exchange were performed continuously (continuous diafiltration) or intermittently (discontinuous diafiltration), and at least 4 times, preferably 6 times to 10 times or more, more preferably with respect to the starting volume. This was performed using a buffer solution having a volume of 12 times or more.
- the exosomes isolated in Example 2 were measured for particle size and concentration by nanoparticle tracking analysis ( NTA; purchased from Malvern).
- NTA nanoparticle tracking analysis
- the NTA analysis results of the exosomes isolated according to the separation method of one embodiment of the present invention are shown in FIGS. 1A and 1B.
- the exosomes obtained after pretreatment and culturing of stem cells in a medium containing ascorbic acid according to an embodiment of the present invention (hereinafter, referred to as “ascorbic acid-treated exosomes") 1A) It can be seen that the particle size distribution is uniform compared to the exosomes obtained after culturing the stem cells in a medium not containing ascorbic acid (hereinafter, referred to as “untreated control exosomes”; FIG. 1B). there was.
- the present invention can produce exosomes having a uniform particle size distribution by pre-treating and culturing stem cells in a medium containing ascorbic acid.
- RAW 264.7 cells were suspended in DMEM medium containing 10% FBS and aliquoted to each well of a multiwell plate to have a confluency of 80 to 90%. The next day, exosomes (each of ascorbic acid-treated exosomes and untreated control exosomes) diluted in fresh medium containing LPS were treated and cultured at an appropriate concentration for 24 hours. At this time, in consideration of the increase in the productivity of exosomes by ascorbic acid, the ascorbic acid-treated exosomes and the untreated control exosomes were adjusted to the same concentration (1.5 ⁇ 10 9 particles/mL) and treated in RAW 264.7 cells.
- the culture supernatant was taken and the inflammatory response was confirmed by measuring the inflammatory cytokines present in the culture supernatant.
- the amount of inflammatory cytokines in the culture supernatant was measured using an IL-6 ELISA kit.
- ELISA kit purchased from R&D system
- the production amount of IL-6 (inflammatory cytokine) in the treated group was confirmed.
- ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, an analog or derivative thereof according to the present invention are anti-inflammatory, wound treatment or wound treatment promoting efficacy It can be seen that it is superior to the treatment control exosomes. Therefore, it can be seen that the method of producing exosomes with enhanced physiological activity or the method of enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.
- the ascorbic acid-treated exosome with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention may be usefully utilized as an active ingredient of a pharmaceutical composition for anti-inflammatory, wound treatment or wound treatment promotion.
- Human skin fibroblasts purchased from Cell Bio Co., Ltd.
- DMEM medium containing fetal bovine serum were dispensed in a multi-well plate, cultured for 24 hours, and cultured for an additional 24 hours in serum-free medium.
- each of the ascorbic acid-treated exosomes and the untreated control exosomes were diluted in serum-free medium at different concentrations, treated with human skin fibroblasts, and cultured for 24 hours at 37° C., 5% CO 2 in an incubator. .
- the ascorbic acid-treated exosomes and the untreated control exosomes were adjusted to the same concentration and treated with human skin fibroblasts.
- each of the ascorbic acid-treated exosome and the untreated control exosome was treated in human skin fibroblasts at a concentration of 5 ⁇ 10 8 particles/mL, and when treated at a high concentration, ascorbic acid-treated exo
- Each of the exosomes and untreated control exosomes was treated with human skin fibroblasts at a concentration of 1.5 ⁇ 10 9 particles/mL.
- the culture supernatant of human skin fibroblasts was collected and centrifuged to prepare a centrifuged culture solution.
- the amount of collagen synthesized from human skin fibroblasts and accumulated in the culture medium was measured using an EIA kit for procollagen type IC-peptide (PIP) (purchased from Takara).
- the total protein amount in the cell lysate was measured with a BCA protein quantification kit (purchased from ThermoFisher Scientific). The measured collagen amount was divided by the total protein amount and normalized to determine the relative collagen amount.
- ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, an analog or derivative thereof according to the present invention are superior to the untreated control exosomes in terms of collagen synthesis increasing efficacy.
- the method of producing exosomes with enhanced physiological activity or the method of enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.
- the ascorbic acid-treated exosomes with enhanced collagen synthesis increasing efficacy of the present invention are superior to the untreated control exosomes in enhancing the physiological activity related to the increase in collagen synthesis, for example, improving skin elasticity, improving wrinkles and/or skin regeneration. And, it can be usefully used as an active ingredient in a cosmetic composition for skin elasticity improvement, wrinkle improvement and/or skin regeneration.
- Example 6 Evaluation of human skin fibroblast proliferation efficacy of exosomes
- Human skin fibroblasts (purchased from Cell Bio Co., Ltd.) dispersed in DMEM medium containing fetal bovine serum were dispensed in a multi-well plate, cultured for 24 hours, and cultured for an additional 24 hours in serum-free medium. Thereafter, each of the ascorbic acid-treated exosomes and the untreated control exosomes and Nuclight reagent (purchased from Sartorius) were diluted in serum-free medium, treated with human skin fibroblasts, and human skin fibroblasts were incubated at 37°C, 5% CO 2 incubated for 72 hours.
- ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, analogs or derivatives thereof according to the present invention are non-treated control exosomes in terms of the proliferation promoting efficacy of human skin fibroblasts.
- the ascorbic acid-treated exosome of the present invention enhances physiological activity related to promotion of human skin fibroblast proliferation, for example, skin elasticity improvement, wrinkle improvement, skin regeneration, wound treatment and/or wound treatment promotion, than the untreated control exosome. Efficacy is excellent. Therefore, it can be seen that the method of producing exosomes with enhanced physiological activity or the method of enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.
- the ascorbic acid-treated exosome of the present invention can be usefully used as an active ingredient in a cosmetic composition for skin elasticity improvement, wrinkle improvement and/or skin regeneration, and a pharmaceutical composition for wound treatment or wound treatment promotion.
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Abstract
The present invention provides a method for enhancing the biological activity of exosomes, comprising a step of culturing animal-derived cells in a medium containing ascorbic acid, or an analogue or derivative thereof. A method for producing exosomes having enhanced biological activity or a method for enhancing the biological activity of exosomes, of the present invention, enables the production of commercially and/or clinically useful exosomes having enhanced biological activity, and the exosomes having enhanced biological activity, produced thereby, can be effectively used as an active ingredient of a pharmaceutical composition or a cosmetic composition.
Description
본 발명은 엑소좀의 생산방법에 관한 것으로서, 생리활성이 강화된 엑소좀의 생산방법에 관한 것이다.The present invention relates to a method for producing exosomes, and to a method for producing exosomes with enhanced physiological activity.
또한, 본 발명은 엑소좀의 생리활성 강화 방법, 그리고 이를 위한 배지에 관한 것이다.In addition, the present invention relates to a method for enhancing the physiological activity of exosomes, and a medium for the same.
추가로, 본 발명은 전술한 바와 같은 생산방법에 의해 수득된 생리활성이 강화된 엑소좀의 응용에 관한 것이다.Further, the present invention relates to the application of the exosomes with enhanced physiological activity obtained by the production method as described above.
최근 세포 분비물(secretome)에 세포의 행동(behavior)을 조절하는 다양한 생체활성인자가 포함되어 있다는 연구가 보고되고 있으며, 특히 세포 분비물 내에는 세포 간 신호전달 기능을 갖는 '엑소좀(exosome)' 또는 '세포외 소포체(extracellular vesicle)'가 포함되어 있어 그 성분과 기능에 대한 연구가 활발히 진행 중에 있다. Recently, it has been reported that cell secretome contains various bioactive factors that regulate cell behavior. In particular, cell secretion contains 'exosome' or Because it contains 'extracellular vesicle', research on its components and functions is being actively conducted.
세포는 세포외 환경에 다양한 막(membrane) 유형의 소포체를 방출하는데, 통상 이러한 방출 소포체들을 세포외 소포체(Extracellular vesicles, EVs)라고 부르고 있다. 세포외 소포체는 세포막 유래 소포체, 엑토좀(ectosomes), 쉐딩 소포체(shedding vesicles), 마이크로파티클(microparticles), 엑소좀 등으로 불려지기도 하며, 경우에 따라서는 엑소좀과는 구별되어 사용되기도 한다.Cells release various membrane types of ERs to the extracellular environment, and these ERs are commonly referred to as extracellular vesicles (EVs). The extracellular vesicles are also called cell membrane-derived ERs, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, they are used separately from exosomes.
엑소좀은 세포막의 구조와 동일한 이중인지질막으로 이루어진 수십 내지 수백 나노미터 크기의 소포체로서 내부에는 엑소좀 카고(cargo)라고 불리는 단백질, 핵산(mRNA, miRNA 등) 등이 포함되어 있다. 엑소좀 카고에는 광범위한 신호전달 요소들(signaling factors)이 포함되며, 이들 신호전달 요소들은 세포 타입에 특이적이고 분비세포의 환경에 따라 상이하게 조절되는 것으로 알려져 있다. 엑소좀은 세포가 분비하는 세포 간 신호전달 매개체로서 이를 통해 전달된 다양한 세포 신호는 표적 세포의 활성화, 성장, 이동, 분화, 탈분화, 사멸(apoptosis), 괴사(necrosis)를 포함한 세포 행동을 조절한다고 알려져 있다. 엑소좀은 유래된 세포의 성질 및 상태에 따라 특이적인 유전물질과 생체활성 인자들이 포함되어 있다. 증식하는 줄기세포 유래 엑소좀의 경우 세포의 이동, 증식 및 분화와 같은 세포 행동을 조절하고, 조직 재생과 관련된 줄기세포의 특성이 반영되어 있다(Nature Review Immunology 2002 (2) 569-579).Exosomes are endoplasmic reticulum with a size of several tens to hundreds of nanometers made of a double phospholipid membrane identical to the structure of the cell membrane, and the inside contains proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known Exosomes contain specific genetic material and bioactive factors according to the nature and state of the cell from which they are derived. In the case of proliferating stem cell-derived exosomes, it regulates cell behaviors such as cell migration, proliferation and differentiation, and reflects the characteristics of stem cells related to tissue regeneration (Nature Review Immunology 2002 (2) 569-579).
즉, 세포의 아바타라고 불리는 엑소좀은 세포와 유사하게 성장인자와 같은 생체활성 인자들이 포함되어 있는데, 생체활성 인자들을 세포와 세포 간에 실어 나르는 전달체 역할, 즉, 세포와 세포 간의 교신 역할을 한다. 엑소좀은 줄기세포, 면역세포, 섬유아세포 및 암세포 등의 동물세포로부터 방출될 뿐만 아니라 식물, 세균(bacteria), 균류(fungi), 조류(algae) 등 다양한 생물의 세포로부터도 방출되는 것으로 알려져 있다. That is, exosomes called avatars of cells contain bioactive factors such as growth factors similar to cells, and serve as a carrier for transporting bioactive factors between cells, that is, communication between cells. Exosomes are known not only to be released from animal cells such as stem cells, immune cells, fibroblasts and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae. .
엑소좀을 분리하는 종래 기술로는 초원심분리법(ultracentrifugation), 밀도구배원심법(density gradient centrifugation), 초미세여과법(ultrafiltration), 접선흐름여과법(Tangential Flow Filtration; TFF), 크기 배제 크로마토그래피(size exclusion chromatography), 이온교환 크로마토그래피(ion exchange chromatography), 면역친화성 분리법(immunoaffinity capture), 미세유체기술 분리법(microfluidics-based isolation), 침전법(exosome precipitation), 총엑소좀 추출 키트(total exosome isolation kit), 또는 폴리머 기반 침전법(polymer based precipitation) 등이 있다.Conventional techniques for separating exosomes include ultracentrifugation, density gradient centrifugation, ultrafiltration, tangential flow filtration (TFF), size exclusion chromatography (size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit), or polymer based precipitation.
그러나, 전술한 바와 같이 다양한 엑소좀 분리방법이 제안되고 있음에도 불구하고 다른 기술분야와 마찬가지로 엑소좀의 생산과 관련한 기술분야에서도 끊임없는 개선이 이루어질 수 있는 것이다. 예를 들어, 엑소좀의 생리활성 인자들과 관련된 효능을 향상시킬 수 있는 새로운 엑소좀 생산기술의 제공이 필요하다.However, as described above, although various exosome separation methods have been proposed, continuous improvement can be made in the technical field related to the production of exosomes as in other technical fields. For example, it is necessary to provide a new exosome production technology that can improve the efficacy related to the physiologically active factors of the exosome.
한편, 상기한 배경기술로서 설명된 사항들은 본 발명의 배경에 대한 이해 증진을 위한 것일 뿐, 본 발명의 "선행 기술"로서 이용될 수 있다는 승인으로서 인용한 것은 아님을 이해하여야 한다.On the other hand, it should be understood that the matters described as the above background art are only for improving understanding of the background of the present invention, and are not cited as approval that they can be used as "prior art" of the present invention.
본 발명의 목적은 생리활성이 강화된 엑소좀의 생산방법을 제공하는데 있다.It is an object of the present invention to provide a method for producing exosomes with enhanced physiological activity.
본 발명의 다른 목적은 엑소좀의 생리활성 강화 방법을 제공하는데 있다.Another object of the present invention is to provide a method for enhancing the physiological activity of exosomes.
본 발명의 또 다른 목적은 엑소좀의 생리활성 강화용 배지를 제공하는데 있다.Another object of the present invention is to provide a medium for enhancing the physiological activity of exosomes.
본 발명의 또 다른 목적은 전술한 바와 같은 생산방법에 의해 수득된 생리활성이 강화된 엑소좀의 다양한 응용을 제공하는데 있다.Another object of the present invention is to provide various applications of the exosomes with enhanced physiological activity obtained by the production method as described above.
그러나, 전술한 바와 같은 본 발명의 과제는 예시적인 것으로, 이에 의해 본 발명의 범위가 제한되는 것은 아니다. 또한, 본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.However, the problems of the present invention as described above are exemplary, and the scope of the present invention is not limited thereto. Further, other objects and advantages of the present invention will become more apparent by the following detailed description of the invention, claims and drawings.
본 발명자들은 생리활성이 강화된 엑소좀의 생산방법에 대해 예의 연구를 거듭하던 중, 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 세포를 배양할 경우 배양액으로부터 분리된 엑소좀의 생리활성이 향상되는 것을 확인하여 본 발명을 완성하였다.The present inventors have been repeating intensive studies on a method for producing exosomes with enhanced physiological activity, and when cells are cultured in a medium containing ascorbic acid, an analog or derivative thereof, the physiological activity of the exosomes isolated from the culture medium is improved It was confirmed that the present invention was completed.
본 명세서에서 용어, "세포외 소포체(Extracellular vesicles, EVs)"는 통상 세포막 유래 소포체(membrane vesicles), 엑토좀(ectosomes), 쉐딩 소포체(shedding vesicles), 마이크로파티클(microparticles), 또는 이의 등가물을 포괄하는 의미로 사용된다. 분리 환경, 조건 및 방법 등에 따라 세포외 소포체는 엑소좀과 동일한 의미를 가질 수도 있고 엑소좀과 크기는 동일 내지는 유사하지만 엑소좀의 조성을 갖지 않는 나노베지클까지 포함하는 의미를 가질 수도 있다. 한편, 본 명세서에서 사용되는 엑소좀이라는 용어는 상기 세포외 소포체를 포괄하는 의미로 사용된다.As used herein, the term "extracellular vesicles (EVs)" generally encompasses membrane vesicles, ectosomes, shedding vesicles, microparticles, or equivalents thereof. used in the sense that Depending on the isolation environment, conditions and methods, etc., the extracellular vesicle may have the same meaning as an exosome, and may have the same or similar size as the exosome, but may also have a meaning including nanovesicles that do not have the composition of the exosome. On the other hand, the term exosome used herein is used to encompass the extracellular vesicles.
본 명세서에서 용어, "엑소좀(exosomes)"은 세포막의 구조와 동일한 이중인지질막으로 이루어진 수십 내지 수백 나노미터(바람직하게는 대략 30~200 nm) 크기의 소포체를 의미한다(단, 분리 대상이 되는 세포 종류, 분리방법 및 측정방법에 따라 엑소좀의 입자 크기는 가변될 수 있음)(Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015) DOI 10.1007/s00216-015-8535-3). 엑소좀에는 엑소좀 카고(cargo)라고 불리는 단백질, 핵산(mRNA, miRNA 등) 등이 포함되어 있다. 엑소좀 카고에는 광범위한 신호전달 요소들(signaling factors)이 포함되며, 이들 신호전달 요소들은 세포 타입에 특이적이고 분비세포의 환경에 따라 상이하게 조절되는 것으로 알려져 있다. 엑소좀은 세포가 분비하는 세포 간 신호전달 매개체로서 이를 통해 전달된 다양한 세포 신호는 표적 세포의 활성화, 성장, 이동, 분화, 탈분화, 사멸(apoptosis), 괴사(necrosis)를 포함한 세포 행동을 조절한다고 알려져 있다.As used herein, the term "exosomes" refers to endoplasmic reticulum with a size of several tens to hundreds of nanometers (preferably about 30 to 200 nm) consisting of a double phospholipid membrane identical to the structure of the cell membrane (provided that the separation target is The particle size of exosomes may vary depending on the type of cell to be used, separation method and measurement method) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015)) DOI 10.1007/s00216-015-8535-3). Exosomes contain proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known
한편, 본 명세서에서 사용된 "엑소좀"이란 용어는 동물 유래 세포에서 분비되어 세포외 공간으로 방출된 나노 크기의 베지클 구조를 갖고 있고 엑소좀과 유사한 조성을 갖는 베지클(예를 들어, 엑소좀-유사 베지클)을 모두 포함하는 것을 의미한다. On the other hand, the term "exosome" used herein has a nano-sized vesicle structure secreted from animal-derived cells and released into the extracellular space and has a composition similar to that of exosomes (eg, exosomes). -It means to include all similar vesicles).
본 발명에 있어서, 엑소좀이 유래하는 동물 유래 세포의 종류는 제한되지 않으나, 본 발명을 한정하지 않는 하나의 예시로서, 줄기세포 또는 면역세포일 수 있다. 상기 줄기세포는 배아줄기세포, 유도만능 줄기세포(induced pluripotent stem cell; iPSC), 성체줄기세포, 배아줄기세포 유래 중간엽 줄기세포, 또는 유도만능 줄기세포 유래 중간엽 줄기세포일 수 있다. 상기 면역세포는 T 세포, B 세포, NK 세포, 세포독성 T 세포(cytotoxic T cell), 수지상 세포 또는 대식 세포일 수 있다. In the present invention, the type of animal-derived cells from which the exosomes are derived is not limited, but may be stem cells or immune cells as an example not limiting the present invention. The stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, embryonic stem cell-derived mesenchymal stem cells, or induced pluripotent stem cell-derived mesenchymal stem cells. The immune cells may be T cells, B cells, NK cells, cytotoxic T cells, dendritic cells or macrophages.
본 발명을 한정하지 않는 예시로서, 상기 성체줄기세포는 중간엽 줄기세포, 인간 조직 유래 중간엽 기질세포, 인간 조직 유래 중간엽 줄기세포, 및 다분화능 줄기세포로 구성된 군에서 선택되는 1종 이상의 성체 줄기세포일 수 있다. 상기 중간엽 줄기세포는 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막, 와튼젤리 및 태반으로 구성된 군에서 선택되는 1종 이상의 조직으로부터 유래된 중간엽 줄기세포일 수 있다. 바람직하게는 상기 성체줄기세포는 중간엽 줄기세포, 예를 들어 지방, 골수, 제대 또는 제대혈 유래 줄기세포일 수 있으며, 보다 바람직하게는 지방 유래 줄기세포일 수 있다. 상기 줄기세포 또는 상기 면역세포의 종류는 병원체에 의한 감염의 위험이 없고 면역 거부 반응을 일으키지 않는 것이라면 제한되지 않으나, 바람직하게는 인간 유래 줄기세포 또는 인간 유래 면역세포일 수 있다.As an example not limiting the present invention, the adult stem cells include one or more adult stem cells selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and pluripotent stem cells. It may be a stem cell. The mesenchymal stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton's jelly and placenta. Preferably, the adult stem cells may be mesenchymal stem cells, for example, stem cells derived from fat, bone marrow, umbilical cord or umbilical cord blood, and more preferably, adipose-derived stem cells. The type of the stem cell or the immune cell is not limited as long as there is no risk of infection by a pathogen and does not cause immune rejection, but preferably a human stem cell or human-derived immune cell.
그러나 인체에 불리한 작용을 일으키지 않는 것이라면 당업계에서 사용되고 있거나 향후 사용될 수 있는 다양한 동물세포를 사용할 수 있음은 물론이다. 예를 들어, HEK 293 세포나 HEK 293T 세포의 사용도 가능하다. 따라서, 후술하는 실시예들에서 사용된 지방줄기세포는 본 발명에서 사용될 수 있는 동물세포의 일례로서 이해되어야 하며, 본 발명은 이에 제한되는 것이 아님을 명백히 밝혀 둔다.However, as long as it does not cause adverse effects on the human body, it is of course possible to use various animal cells that are used in the art or that can be used in the future. For example, it is also possible to use HEK 293 cells or HEK 293T cells. Therefore, the adipose stem cells used in the examples described below should be understood as an example of animal cells that can be used in the present invention, and the present invention is not limited thereto.
본 명세서에서 용어, "아스코르브산, 이의 유사체 또는 유도체"는, 예를 들어, 아스코르브산(Ascorbic Acid), 아스코르브산-2-글루코사이드(Ascorbic acid-2-glucoside; AA-2G), 아스코르브산-2-설페이트(Ascorbic acid-2-sulfate; AA-2S), 아스코르브산-2-포스페이트(Ascorbic acid-2-phosphate; AA-2P), 마그네슘 아스코르빌 포스페이트(Magnesium ascorbyl phosphate), 아스코르빌 팔미테이트 (Ascorbyl palmitate), 레티닐 아스코르브산염 (Retinyl Ascorbate), 테트라헥실데실 아스코르브산염(Tetrahexyldecyl Ascorbate), 아스코르브산 나트륨(Sodium ascorbate), 아스코르브산 칼슘(Calcium ascorbate), 폴리에톡실레이티드 아스코르브산(Polyethoxylated ascorbic acid), 에틸 아스코르브산(Ethyl Ascorbic Acid), 아미노프로필 아스코르빌 포스페이트(Aminopropyl Ascorbyl Phosphate), 및/또는 3-아스코르빌 카르보닐 디펩티드-17(3-Ascorbyl Carbonyl Dipeptide-17)일 수 있다. 그러나 전술한 아스코르브산, 이의 유사체 또는 유도체는 본 발명에서 사용될 수 있는 아스코르브산, 이의 유사체 또는 유도체의 일례로서 이해되어야 하며, 본 발명은 이에 제한되는 것이 아님을 명백히 밝혀 둔다.As used herein, the term "ascorbic acid, an analog or derivative thereof", for example, ascorbic acid (Ascorbic Acid), ascorbic acid-2-glucoside (Ascorbic acid-2-glucoside; AA-2G), ascorbic acid-2 -Sulphate (Ascorbic acid-2-sulfate; AA-2S), ascorbic acid-2-phosphate (AA-2P), magnesium ascorbyl phosphate (Magnesium ascorbyl phosphate), ascorbyl palmitate (Ascorbyl palmitate), Retinyl Ascorbate (Retinyl Ascorbate), Tetrahexyldecyl Ascorbate (Tetrahexyldecyl Ascorbate), Sodium ascorbate (Sodium ascorbate), Calcium ascorbate (Calcium ascorbate), Polyethoxylated ascorbate (Polyethoxylated ascorbate) acid), Ethyl Ascorbic Acid, Aminopropyl Ascorbyl Phosphate, and/or 3-Ascorbyl Carbonyl Dipeptide-17. . However, the above-described ascorbic acid, analogs or derivatives thereof should be understood as examples of ascorbic acid, analogs or derivatives thereof that can be used in the present invention, and the present invention is not limited thereto.
본 명세서에서 용어, "전처리"는 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 동물 유래 세포를 배양하는 것을 의미하고 "전처리 엑소좀 또는 아스코르브산 처리 엑소좀"은 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 동물 유래 세포를 배양하여 얻은 엑소좀을 의미한다. "무처리"란 아스코르브산, 이의 유사체 또는 유도체를 포함하지 않는 배지에서 동물 유래 세포를 배양하는 것을 의미하고, "무처리 엑소좀, 무처리 대조군 엑소좀 또는 무처리 대조군"은 세포배양 단계 및 엑소좀 분리 단계 중 어떠한 단계에서도 아스코르브산, 이의 유사체 또는 유도체가 처리되지 않고 수득된 엑소좀을 의미한다. As used herein, the term "pretreatment" refers to culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and "pretreatment exosome or ascorbic acid-treated exosome" refers to ascorbic acid, an analog or derivative thereof. It refers to an exosome obtained by culturing animal-derived cells in a containing medium. "Untreated" means culturing animal-derived cells in a medium that does not contain ascorbic acid, an analog or derivative thereof, and "untreated exosomes, untreated control exosomes or untreated control" refers to the cell culture step and exo It refers to exosomes obtained without treatment with ascorbic acid, an analog or a derivative thereof, in any step of the isolation step.
본 명세서에서 용어, "항염"이란 염증을 예방, 억제, 완화, 개선 또는 치료하는 것을 의미하고, 염증성 질환으로는 본 발명을 제한하지 않는 예시로서, 피부염, 아토피성 피부염, 습진, 박테리아 감염, 바이러스 감염 또는 곰팡이 감염으로 인한 염증, 화상, 화상으로 인한 염증, 상처, 상처로 인한 염증, 궤양성 대장염, 관절염, 류머티스성 관절염, 간염, 신장염 등을 들 수 있다. As used herein, the term "anti-inflammatory" means preventing, inhibiting, alleviating, ameliorating or treating inflammation, and inflammatory diseases are examples that are not limited to the present invention, and include dermatitis, atopic dermatitis, eczema, bacterial infection, virus Inflammation due to infection or fungal infection, burns, inflammation due to burns, wounds, inflammation due to wounds, ulcerative colitis, arthritis, rheumatoid arthritis, hepatitis, nephritis, and the like.
본 명세서에서 사용된 용어 "창상(wound)"은 생체가 손상된 상태를 의미하며, 생체 내부 또는 외부 표면을 이루는 조직, 예를 들면 피부, 근육, 신경조직, 뼈, 연조직, 내부기관 또는 혈관조직이 분단 또는 파괴된 병리학적 상태를 포괄한다. 본 발명을 한정하지 않는 예시로서 창상은 찰과상(abrasion), 열상(laceration), 자상, 절상, 결출상(avulsion), 욕창, 와창, 방사선 조사에 의한 조직의 파괴, 관통상(penetrated wound), 총상(gun shot wound), 화상, 동상, 수술상, 성형수술 후 봉합 부위, 화학적 창상 등을 포함하며 개체의 어떠한 부분에 대한 손상이 포함될 수 있다.As used herein, the term "wound" refers to a state in which a living body is damaged, and a tissue constituting the internal or external surface of the living body, for example, skin, muscle, nerve tissue, bone, soft tissue, internal organ, or vascular tissue It encompasses segmented or disrupted pathological conditions. As an example not limiting the present invention, the wound is abrasion, laceration, cut, cut, avulsion, pressure sore, pit wound, tissue destruction by radiation, penetrated wound, gunshot wound ( gun shot wound), burns, frostbite, surgical wounds, sutures after plastic surgery, chemical wounds, etc., and may include damage to any part of an object.
본 명세서에서 용어, "이온토포레시스(iontophoresis)"는 유효물질이 적용된 피부에 미세전류를 흐르게 하여 전위차를 주어 피부의 전기적 환경을 변화시킴으로써 이온화된 유효성분을 전기적 반발력으로 피부를 투과하게 하는 방법을 의미한다. 본 발명의 일 구체예에 사용되는 이온토포레시스(iontophoresis)는 피부 위의 전극 패치에 외부전원으로부터의 전류가 흘러들어가 피부에 미세전류가 도입되는 방식, 전극 패치 자체에 배터리가 장착되어 피부에 미세전류가 도입되는 방식, 고농도 전해질 용액 및 저농도 전해질 용액 간의 이온 농도 차이를 통해 전류를 발생시키는 역전기투석(Reversed Electrodialysis) 수단이 장착된 패치를 통해 피부에 미세전류가 도입되는 방식 등을 포함할 수 있다. 그러나, 본 발명은 이에 제한되는 것이 아니며, 다양한 방식의 이온토포레시스가 사용될 수 있음은 물론이다.As used herein, the term "iontophoresis" refers to a method of allowing an ionized active ingredient to permeate the skin with an electrical repulsive force by flowing a microcurrent to the skin to which an active material is applied to change the electrical environment of the skin by giving a potential difference means Iontophoresis used in one embodiment of the present invention is a method in which a current from an external power source flows into an electrode patch on the skin to introduce a microcurrent to the skin, and a battery is mounted on the electrode patch itself to give the skin A method in which microcurrent is introduced, a method in which microcurrent is introduced into the skin through a patch equipped with a reversed electrodialysis means that generates an electric current through a difference in ion concentration between a high-concentration electrolyte solution and a low-concentration electrolyte solution, etc. can However, the present invention is not limited thereto, and it goes without saying that various types of iontophoresis may be used.
본 발명은 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하는 단계를 포함하는, 엑소좀의 생리활성 강화 방법을 제공한다.The present invention provides a method for enhancing the physiological activity of exosomes, comprising culturing an animal-derived cell in a medium containing ascorbic acid, an analog or derivative thereof.
본 발명의 일 구체예의 엑소좀의 생리활성 강화 방법은 상기 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 무혈청 배지에서 추가로 배양하는 단계를 포함할 수 있다. The method for enhancing the physiological activity of an exosome of one embodiment of the present invention may include further culturing the animal-derived cells in a serum-free medium containing ascorbic acid, an analog or a derivative thereof.
본 발명의 일 구체예의 엑소좀의 생리활성 강화 방법에 있어서, 상기 아스코르브산, 이의 유사체 또는 유도체의 농도는 약 1 μg/mL 내지 300 μg/mL, 바람직하게는 약 10 μg/mL 내지 100 μg/mL일 수 있다.In the method of enhancing the physiological activity of an exosome of one embodiment of the present invention, the concentration of the ascorbic acid, an analog or derivative thereof is about 1 μg/mL to 300 μg/mL, preferably about 10 μg/mL to 100 μg/mL mL.
본 발명의 일 구체예의 엑소좀의 생리활성 강화 방법에 있어서, 상기 생리활성 강화는 항염, 창상 치료 또는 창상 치료 촉진 효능 강화일 수 있다. 또한, 상기 생리활성 강화는 콜라겐 합성 증가 효능 강화일 수 있고, 예를 들어 피부탄력개선 강화, 주름개선 강화 또는 피부재생 강화 중 적어도 하나일 수 있다.In the method of enhancing the physiological activity of the exosome of one embodiment of the present invention, the enhancement of the physiological activity may be anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancement. In addition, the enhancement of the physiological activity may be enhancement of the effect of increasing collagen synthesis, for example, may be at least one of skin elasticity enhancement enhancement, wrinkle improvement enhancement enhancement, or skin regeneration enhancement enhancement.
본 발명은 아스코르브산, 이의 유사체 또는 유도체를 포함하는, 동물세포 유래의 엑소좀의 생리활성 강화용 배지를 제공한다. 상기 생리활성 강화는 항염, 창상 치료 또는 창상 치료 촉진 효능 강화일 수 있다. 또한, 상기 생리활성 강화는 콜라겐 합성 증가 효능 강화일 수 있고, 예를 들어 피부탄력개선 강화, 주름개선 강화 또는 피부재생 강화 중 적어도 하나일 수 있다. The present invention provides a medium for enhancing the physiological activity of exosomes derived from animal cells, comprising ascorbic acid, an analog or derivative thereof. The enhancement of the physiological activity may be anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancement. In addition, the enhancement of the physiological activity may be enhancement of the effect of increasing collagen synthesis, for example, may be at least one of skin elasticity enhancement enhancement, wrinkle improvement enhancement enhancement, or skin regeneration enhancement enhancement.
[규칙 제91조에 의한 정정 07.03.2022]
본 발명의 일 구체예의 동물세포 유래의 엑소좀의 생리활성 강화용 배지에 있어서, 상기 아스코르브산, 이의 유사체 또는 유도체의 농도는 약 1 μg/mL 내지 300 μg/mL, 바람직하게는 약 10 μg/mL 내지 100 μg/mL일 수 있다.[Correction by Rule 91 07.03.2022]
In the medium for enhancing the physiological activity of animal cell-derived exosomes of one embodiment of the present invention, the concentration of the ascorbic acid, an analog or derivative thereof is about 1 μg/mL to 300 μg/mL, preferably about 10 μg/mL mL to 100 μg/mL.
본 발명의 일 구체예의 동물세포 유래의 엑소좀의 생리활성 강화용 배지에 있어서, 상기 아스코르브산, 이의 유사체 또는 유도체의 농도는 약 1 μg/mL 내지 300 μg/mL, 바람직하게는 약 10 μg/mL 내지 100 μg/mL일 수 있다.[Correction by Rule 91 07.03.2022]
In the medium for enhancing the physiological activity of animal cell-derived exosomes of one embodiment of the present invention, the concentration of the ascorbic acid, an analog or derivative thereof is about 1 μg/mL to 300 μg/mL, preferably about 10 μg/mL mL to 100 μg/mL.
또한, 본 발명은 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하여 수득된, 생리활성이 강화된 엑소좀을 포함하는 약학 조성물을 제공한다. 예를 들어, 상기 생리활성이 강화된 엑소좀은 항염, 창상 치료 또는 창상 치료 촉진 효능이 강화된 엑소좀일 수 있고, 상기 약학 조성물은 상기 생리활성이 강화된 엑소좀을 유효성분으로 포함하는, 항염, 창상 치료 또는 창상 치료 촉진용 약학 조성물일 수 있다.In addition, the present invention provides a pharmaceutical composition comprising exosomes with enhanced physiological activity obtained by culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof. For example, the exosomes with enhanced physiological activity may be exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy, and the pharmaceutical composition includes the exosomes with enhanced physiological activity as an active ingredient, anti-inflammatory , it may be a pharmaceutical composition for treating wounds or promoting wound healing.
예를 들어, 본 발명의 일 구체예의 약학 조성물은, (a) 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하는 단계와, (b) 상기 동물 유래 세포로부터 분비 또는 방출된 엑소좀을 함유하는 배양액(conditioned media)을 수집한 후 수집한 배양액으로부터 엑소좀을 분리하는 단계를 수행하여 수득된, 항염, 창상 치료 또는 창상 치료 촉진 효능이 강화된 엑소좀을 포함한다.For example, the pharmaceutical composition of one embodiment of the present invention comprises the steps of (a) culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and (b) secreted or released from the animal-derived cells. It includes exosomes with enhanced anti-inflammatory, wound treatment or wound healing promoting efficacy, obtained by performing the step of separating the exosomes from the collected culture medium after collecting the conditioned media containing the exosomes.
[규칙 제91조에 의한 정정 07.03.2022]
본 발명의 일 구체예의 약학 조성물에 있어서, 상기 아스코르브산, 이의 유사체 또는 유도체는 상기 배지 내에 약 1 μg/mL 내지 300 μg/mL의 농도, 바람직하게는 약 10 μg/mL 내지 100 μg/mL의 농도로 포함될 수 있다.[Correction by Rule 91 07.03.2022]
In the pharmaceutical composition of one embodiment of the present invention, the ascorbic acid, analog or derivative thereof is present in the medium at a concentration of about 1 μg/mL to 300 μg/mL, preferably about 10 μg/mL to 100 μg/mL. concentration may be included.
본 발명의 일 구체예의 약학 조성물에 있어서, 상기 아스코르브산, 이의 유사체 또는 유도체는 상기 배지 내에 약 1 μg/mL 내지 300 μg/mL의 농도, 바람직하게는 약 10 μg/mL 내지 100 μg/mL의 농도로 포함될 수 있다.[Correction by Rule 91 07.03.2022]
In the pharmaceutical composition of one embodiment of the present invention, the ascorbic acid, analog or derivative thereof is present in the medium at a concentration of about 1 μg/mL to 300 μg/mL, preferably about 10 μg/mL to 100 μg/mL. concentration may be included.
본 발명을 한정하지 않는 예시로서, 본 발명의 일 구체예의 약학 조성물은 주사, 마이크로니들링, 이온토포레시스, 도포, 또는 이들의 조합에 의해 투여 또는 처리될 수 있다. 예를 들어, 상기 약학 조성물은 주사제형, 주입제형, 분무제형, 액상제형 또는 패취제형일 수 있다. By way of example and not limitation of the present invention, the pharmaceutical composition of one embodiment of the present invention may be administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof. For example, the pharmaceutical composition may be in the form of an injection, injection, spray, liquid or patch.
본 발명의 일 구체예의 약학 조성물은 약학적으로 허용 가능한 담체, 부형제 또는 희석제 등을 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 트레할로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 카보네이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스(microcrystalline cellulose), 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 미네랄 오일 등을 들 수 있으며, 이에 제한되지 않는다. The pharmaceutical composition of one embodiment of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent. The carrier, excipient and diluent include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto. .
본 발명의 일 구체예의 약학 조성물의 투여는 어떠한 적절한 방법으로 환자에게 소정의 물질을 도입하는 것을 의미하며, 상기 약학 조성물의 투여경로는 약물이 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 예를 들어, 본 발명의 일 구체예의 약학 조성물은 경구 또는 비경구 투여할 수 있으며, 비경구 투여로는 관절 내 투여, 관절강 내 투여, 활액낭 내(intrasynovial) 투여, 흉골 내 투여, 수강 내(intrathecal) 투여, 병변 내 및 두개 내(intracranial) 투여, 경피 투여, 복강 내 투여, 정맥 내 투여, 동맥 내 투여, 림프 내 투여, 근육 내 투여, 피하 투여, 피내 투여, 국소 투여, 직장 내 투여 등을 거론할 수 있다. 그러나, 이에 제한되는 것이 아니며 당업계에 알려진 다양한 투여 방법을 배제하지 않는다. 또한, 본 발명의 일 구체예의 약학 조성물은 활성 물질이 표적 조직 또는 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다. 또한, 본 발명의 일 구체예의 약학 조성물의 유효량은 항염, 상처 치유, 및/또는 상처 치유 촉진 효과를 기대하기 위하여 투여에 요구되는 양을 의미한다. Administration of the pharmaceutical composition of one embodiment of the present invention means introducing a predetermined substance to the patient by any suitable method, and the administration route of the pharmaceutical composition is administered through any general route as long as the drug can reach the target tissue. can be For example, the pharmaceutical composition of one embodiment of the present invention may be administered orally or parenterally, and parenteral administration includes intra-articular administration, intra-articular administration, intrasynovial administration, intrasternal administration, intrathecal administration. ) administration, intralesional and intracranial administration, transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intralymphatic administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, rectal administration, etc. can be mentioned However, it is not limited thereto and does not exclude various administration methods known in the art. In addition, the pharmaceutical composition of one embodiment of the present invention can be administered by any device capable of transporting an active substance to a target tissue or cell. In addition, the effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to expect anti-inflammatory, wound healing, and/or wound healing promoting effects.
본 발명의 일 구체예의 약학 조성물의 비경구 투여용 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 또는 좌제일 수 있다. 본 발명의 일 구체예의 약학 조성물의 비경구 투여용 제제는 주사제로도 제조될 수 있다. 본 발명의 일 구체예의 주사제는 수성 주사제, 비수성 주사제, 수성 현탁 주사제, 비수성 현탁 주사제, 또는 용해 또는 현탁하여 사용하는 고형 주사제 등일 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 일 구체예의 주사제는 그 종류에 따라 주사용 증류수, 식물유(예를 들어, 낙화생유, 참기름, 동백기름 등), 모노글리세리드, 디글리세리드, 프로필렌글리콜, 캄퍼, 벤조산에스트라디올, 차살리실산비스무트, 아르세노벤졸나트륨, 또는 황산스트렙토마이신 중 적어도 1종을 포함할 수 있고, 선택적으로 안정제나 방부제를 포함할 수 있다.The formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilized formulation, or a suppository. The formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may also be prepared as an injection. The injection of one embodiment of the present invention may be an aqueous injection, a non-aqueous injection, an aqueous suspension injection, a non-aqueous suspension injection, or a solid injection used by dissolving or suspending, but is not limited thereto. The injection of one embodiment of the present invention is distilled water for injection, vegetable oil (for example, peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, benzoate estradiol, bismuth salicylate, depending on the type , Arsenobenzol sodium, or at least one of streptomycin sulfate, and may optionally include a stabilizer or a preservative.
본 발명의 일 구체예의 약학 조성물의 배합비율은 전술한 바와 같은 추가 성분들의 종류나 양, 형태 등에 따라서 적당하게 선택할 수 있다. 예를 들어, 주사제 전량에 대해, 본 발명의 약학 조성물은 약 0.1 내지 99 중량%, 바람직하게는 약 10 내지 90 중량% 정도 포함될 수 있다. 또한, 본 발명의 일 구체예의 약학 조성물의 적합한 투여량은 질환의 경중, 제형의 종류, 제제화 방법, 환자의 연령, 성별, 체중, 건강 상태, 식이, 배설률, 투여 시간 및 투여 방법에 따라 조절될 수 있다. 예를 들어, 성인에게 본 발명의 일 구체예의 약학 조성물을 투여하는 경우, 하루에 0.001 mg/kg ~ 100 mg/kg의 용량으로 1 내지 수회에 나누어 투여할 수 있다.The blending ratio of the pharmaceutical composition of one embodiment of the present invention can be appropriately selected according to the type, amount, form, etc. of the additional ingredients as described above. For example, based on the total amount of the injection, the pharmaceutical composition of the present invention may be included in about 0.1 to 99% by weight, preferably about 10 to 90% by weight. In addition, a suitable dosage of the pharmaceutical composition of one embodiment of the present invention may be adjusted depending on the severity of the disease, the type of formulation, the formulation method, the patient's age, sex, weight, health status, diet, excretion rate, administration time and administration method. can For example, when the pharmaceutical composition of one embodiment of the present invention is administered to an adult, it may be administered in one to several divided doses at a dose of 0.001 mg/kg to 100 mg/kg per day.
또한, 본 발명은 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하여 수득된, 콜라겐 합성 증가 효능이 강화된 엑소좀을 포함하는 화장료 조성물을 제공한다. 상기 화장료 조성물은 예를 들어 피부탄력개선, 주름개선 및/또는 피부재생용 화장료 조성물일 수 있다. In addition, the present invention provides a cosmetic composition comprising exosomes with enhanced collagen synthesis-increasing efficacy obtained by culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof. The cosmetic composition may be, for example, a cosmetic composition for skin elasticity improvement, wrinkle improvement and/or skin regeneration.
예를 들어, 본 발명의 일 구체예의 화장료 조성물은, (a) 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하는 단계와, (b) 상기 동물 유래 세포로부터 분비 또는 방출된 엑소좀을 함유하는 배양액(conditioned media)을 수집한 후 수집한 배양액으로부터 엑소좀을 분리하는 단계를 수행하여 수득된, 콜라겐 합성 증가 효능이 강화된 엑소좀을 포함한다.For example, the cosmetic composition of one embodiment of the present invention comprises the steps of (a) culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and (b) secreted or released from the animal-derived cells. After collecting the culture medium containing the exosomes (conditioned media), it includes exosomes with enhanced collagen synthesis effect obtained by performing the step of separating the exosomes from the collected culture medium.
[규칙 제91조에 의한 정정 07.03.2022]
본 발명의 일 구체예의 화장료 조성물에 있어서, 상기 아스코르브산, 이의 유사체 또는 유도체는 상기 배지 내에 약 1 μg/mL 내지 300 μg/mL의 농도, 바람직하게는 약 10 μg/mL 내지 100 μg/mL의 농도로 포함될 수 있다.[Correction by Rule 91 07.03.2022]
In the cosmetic composition of one embodiment of the present invention, the ascorbic acid, analog or derivative thereof is present in a concentration of about 1 μg/mL to 300 μg/mL in the medium, preferably about 10 μg/mL to 100 μg/mL. concentration may be included.
본 발명의 일 구체예의 화장료 조성물에 있어서, 상기 아스코르브산, 이의 유사체 또는 유도체는 상기 배지 내에 약 1 μg/mL 내지 300 μg/mL의 농도, 바람직하게는 약 10 μg/mL 내지 100 μg/mL의 농도로 포함될 수 있다.[Correction by Rule 91 07.03.2022]
In the cosmetic composition of one embodiment of the present invention, the ascorbic acid, analog or derivative thereof is present in a concentration of about 1 μg/mL to 300 μg/mL in the medium, preferably about 10 μg/mL to 100 μg/mL. concentration may be included.
예를 들어, 상기 화장료 조성물은 샴푸, 비누, 린스, 계면활성제-함유 클렌징, 크림, 로션, 연고, 토닉, 트리트먼트, 컨디셔너, 현탁액, 유탁액, 페이스트, 젤, 오일, 왁스, 스프레이, 에어졸, 미스트, 또는 파우더일 수 있고, 바람직하게는 로션이나 크림일 수 있다. For example, the cosmetic composition may include shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, It may be a mist or powder, and preferably a lotion or cream.
한편, 본 발명의 일 구체예의 화장료 조성물은 본 발명의 효과를 손상하지 않는 범위내에서 통상 화장료 조성물에 이용되는 성분, 예를 들면 보습제, 산화방지제, 유성성분, 자외선 흡수제, 유화제, 계면활성제, 증점제, 알콜류, 분말성분, 색재, 수성성분, 물, 각종 피부영양제 등을 필요에 따라 적절히 배합할 수 있다. On the other hand, the cosmetic composition of one embodiment of the present invention is a component commonly used in a cosmetic composition within a range that does not impair the effects of the present invention, for example, a moisturizer, an antioxidant, an oily component, a UV absorber, an emulsifier, a surfactant, a thickener , alcohols, powder components, colorants, water-based components, water, and various skin nutrients can be appropriately blended as needed.
또한, 본 발명의 일 구체예의 화장료 조성물은 생리활성이 강화된 엑소좀 이외에, 그 작용(예를 들어, 피부탄력개선, 주름개선 및/ 피부재생 등)을 손상시키지 않는 한도에서 종래부터 사용된 피부탄력개선제, 주름방지제 및/또는 보습제를 함께 혼합하여 사용할 수 있다. 예를 들어, 본 발명의 생리활성이 강화된 엑소좀을 유효성분으로 포함하는 화장료 조성물은 하이드로겔, 히알루론산, 히알루론산 염(예를 들어 히알루론산 나트륨 등), 또는 히알루론산 겔 중 적어도 1종에 담지되거나 혼합될 수 있다. 본 발명의 일 구체예의 화장료 조성물에서, 상기 하이드로겔의 종류는 제한되지 않으나, 바람직하게는 겔화 고분자를 다가 알코올에 분산시켜 얻은 하이드로겔일 수 있다. 상기 겔화 고분자는 플루로닉, 정제한천, 아가로오스, 젤란검, 알긴산, 카라기난, 카시아검, 잔탄검, 갈락토만난, 글루코만난, 펙틴, 셀룰로오스, 구아검 및 로커스트빈검으로 이루어진 군으로부터 선택된 적어도 1종일 수 있고, 상기 다가 알코올은 에틸렌글리콜, 프로필렌글리콜, 1,3-부틸렌글리콜, 이소부틸렌글리콜, 디프로필렌글리콜, 소르비톨, 자일리톨 및 글리세린으로 이루어진 군으로부터 선택된 적어도 1종일 수 있다.In addition, the cosmetic composition of one embodiment of the present invention, in addition to exosomes with enhanced physiological activity, conventionally used skin as long as it does not impair its action (eg, skin elasticity improvement, wrinkle improvement and/or skin regeneration, etc.) An elasticity improving agent, an anti-wrinkle agent, and/or a moisturizing agent may be mixed and used together. For example, the cosmetic composition comprising the exosome with enhanced physiological activity of the present invention as an active ingredient is at least one of hydrogel, hyaluronic acid, hyaluronic acid salt (eg sodium hyaluronate, etc.), or hyaluronic acid gel may be supported or mixed. In the cosmetic composition of one embodiment of the present invention, the type of the hydrogel is not limited, but may preferably be a hydrogel obtained by dispersing a gelling polymer in a polyhydric alcohol. The gelling polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum. and the polyhydric alcohol may be at least one selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol and glycerin.
본 발명의 일 구체예의 화장료 조성물은 예를 들면, 패취, 마스크팩, 마스크시트, 크림, 토닉, 연고, 현탁액, 유탁액, 페이스트, 로션, 젤, 오일, 팩, 스프레이, 에어졸, 미스트, 파운데이션, 파우더, 기름 종이 등의 다양한 형태에 적용할 수 있다. 예를 들어, 상기 화장료 조성물은 패취, 마스크팩 또는 마스크시트의 적어도 일면(一面)에 도포되거나 침적될 수 있다.The cosmetic composition of one embodiment of the present invention is, for example, a patch, mask pack, mask sheet, cream, tonic, ointment, suspension, emulsion, paste, lotion, gel, oil, pack, spray, aerosol, mist, foundation, It can be applied to various forms such as powder and oil paper. For example, the cosmetic composition may be applied or deposited on at least one surface of a patch, mask pack, or mask sheet.
본 발명의 일 구체예의 화장료 조성물은 피부탄력개선, 주름개선 및/또는 피부재생 등의 목적으로 사용될 수 있으며, 화장품 제형은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있다. 예를 들어 패취, 마스크팩, 마스크시트, 유연화장수, 영양화장수, 수렴화장수, 영양크림, 마사지크림, 아이크림, 클렌징크림, 에센스, 아이에센스, 클렌징로션, 클렌징폼, 클렌징워터, 선스크린, 립스틱, 비누, 샴푸, 계면활성제-함유 클렌징, 입욕제, 바디로션, 바디크림, 바디오일, 바디에센스, 바디세정제, 염모제, 헤어토닉 등으로 제형화할 수 있으나 이에 한정되는 것은 아니다.The cosmetic composition of one embodiment of the present invention may be used for the purpose of skin elasticity improvement, wrinkle improvement and/or skin regeneration, and the cosmetic formulation may be prepared in any formulation conventionally prepared in the art. For example, patch, mask pack, mask sheet, flexible lotion, nourishing lotion, astringent lotion, nourishing cream, massage cream, eye cream, cleansing cream, essence, eye essence, cleansing lotion, cleansing foam, cleansing water, sunscreen, lipstick , soap, shampoo, surfactant-containing cleansing, bathing agent, body lotion, body cream, body oil, body essence, body wash, hair dye, hair tonic, etc., but is not limited thereto.
본 발명의 일 구체예의 화장료 조성물은 화장료 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제 그리고 담체를 포함할 수 있다. 또한, 화장료 조성물에 대한 각각의 제형에 있어서, 다른 성분들은 화장료 조성물의 종류 또는 사용목적 등에 따라 당업자가 어려움 없이 적의 선정하여 배합할 수 있다.The cosmetic composition of one embodiment of the present invention includes components commonly used in the cosmetic composition, for example, it may include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. In addition, in each formulation for the cosmetic composition, other components can be appropriately selected and formulated by those skilled in the art without difficulty depending on the type or purpose of use of the cosmetic composition.
본 발명은 엑소좀의 항염, 창상 치료 또는 창상 치료 촉진 효능 내지는 콜라겐 합성과 같은 생리활성을 강화시킬 수 있는 우수한 효과를 나타낸다. 따라서, 본 발명의 생리활성이 강화된 엑소좀의 생산방법 내지는 엑소좀의 생리활성 강화 방법은 상업적 및/또는 임상적으로 유용한 생리활성이 강화된 엑소좀을 생산할 수 있고, 이와 같이 생산된 생리활성이 강화된 엑소좀은 약학 조성물 또는 화장료 조성물의 유효성분으로 유용하게 활용될 수 있다.The present invention exhibits an excellent effect capable of enhancing the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes or physiological activities such as collagen synthesis. Therefore, the method for producing exosomes with enhanced physiological activity or the method for enhancing the physiological activity of exosomes of the present invention can produce commercially and/or clinically useful exosomes with enhanced physiological activity, and thus produced physiological activity This reinforced exosome can be usefully utilized as an active ingredient in a pharmaceutical composition or cosmetic composition.
한편, 전술한 바와 같은 효과들에 의해 본 발명의 범위가 제한되는 것은 아니다.On the other hand, the scope of the present invention is not limited by the above-described effects.
도 1A는 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 줄기세포를 전처리 및 배양한 후 수득된 엑소좀에 대해 NTA (nanoparticle tracking analysis)를 수행하여 얻은 입자 크기 분포와 입자수를 나타내는 그래프이다. 1A is a graph showing the particle size distribution and the number of particles obtained by performing NTA (nanoparticle tracking analysis) on exosomes obtained after pretreatment and culturing of stem cells in a medium containing ascorbic acid, an analog or derivative thereof.
도 1B는 아스코르브산, 이의 유사체 또는 유도체를 포함하지 않는 배지에서 줄기세포를 전처리 및 배양한 후 수득된 엑소좀에 대해 NTA (nanoparticle tracking analysis)를 수행하여 얻은 입자 크기 분포와 입자수를 나타내는 그래프이다.1B is a graph showing the particle size distribution and the number of particles obtained by performing NTA (nanoparticle tracking analysis) on exosomes obtained after pretreatment and culturing of stem cells in a medium not containing ascorbic acid, analogs or derivatives thereof. .
도 2는 본 발명의 일 구체예에 따라 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 줄기세포를 전처리 및 배양한 후 수득된 엑소좀(이하, "아스코르브산 처리 엑소좀"이라 함)의 항염 효능이 아스코르브산, 이의 유사체 또는 유도체를 포함하지 않는 배지에서 줄기세포를 배양한 후 수득된 엑소좀(이하, "무처리 대조군 엑소좀"이라 함)에 비해 현저하게 증가한 것을 나타내는 비교 그래프이다.Figure 2 is an anti-inflammatory of the exosomes obtained after pretreatment and culturing of stem cells in a medium containing ascorbic acid, an analog or derivative thereof according to an embodiment of the present invention (hereinafter, referred to as "ascorbic acid-treated exosomes") It is a comparative graph showing that the efficacy is significantly increased compared to the exosomes obtained after culturing the stem cells in a medium not containing ascorbic acid, analogs or derivatives thereof (hereinafter, referred to as "untreated control exosomes").
도 3은 본 발명의 아스코르브산 처리 엑소좀의 콜라겐 합성 촉진 효능이 무처리 대조군 엑소좀에 비해 현저하게 증가한 것을 나타내는 비교 그래프이다.3 is a comparative graph showing that the collagen synthesis promoting effect of the ascorbic acid-treated exosome of the present invention is significantly increased compared to that of the untreated control exosome.
도 4는 본 발명의 아스코르브산 처리 엑소좀의 세포 증식 촉진 효능이 무처리 대조군 엑소좀에 비해 현저하게 증가한 것을 나타내는 비교 그래프이다.4 is a comparative graph showing that the cell proliferation promoting effect of the ascorbic acid-treated exosomes of the present invention is significantly increased compared to the untreated control exosomes.
이하 본 발명을 하기 실시예에서 보다 상세하게 기술한다. 다만, 하기 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 권리범위를 제한하거나 한정하는 것이 아니다. 본 발명의 상세한 설명 및 실시예로부터 본 발명이 속하는 기술분야의 통상의 기술자가 용이하게 유추할 수 있는 것은 본 발명의 권리범위에 속하는 것으로 해석된다. 본 발명에 인용된 참고문헌들은 본 발명에 참고로서 통합된다.Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or limit the scope of the present invention. What can be easily inferred by those skilled in the art from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention. The references cited herein are incorporated herein by reference.
명세서 전체에서, 어떤 부분이 어떤 구성 요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성 요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.
실시예Example
실시예 1: 세포의 배양Example 1: Cultivation of cells
인간 지방 유래 줄기세포를 10% 우태아혈청 (Fetal bovine serum, FBS), 100 유닛(Units)/mL 페니실린, 및 100 μg/mL 스트렙토마이신이 포함된 DMEM 배양 배지에 현탁시킨 후, 플라스크에 접종하고 37℃, 5% CO2 배양기에서 배양하였다. 아스코르브산 처리군의 경우 배양 배지에 30μg/mL의 아스코르브산을 첨가하였다. Human adipose-derived stem cells were suspended in DMEM culture medium containing 10% fetal bovine serum (FBS), 100 Units/mL penicillin, and 100 μg/mL streptomycin, and then inoculated into a flask. 37° C., 5% CO 2 Incubated in an incubator. In the case of the ascorbic acid-treated group, 30 μg/mL of ascorbic acid was added to the culture medium.
아스코르브산 처리군 및 무처리군 모두 80% 이상의 세포 밀집도(confluency)에 도달하면, 세포를 인산염 완충용액(phosphate-buffered saline; PBS, Thermo Scientific에서 구입)으로 세척 후, 배양배지를 100 유닛/mL 페니실린 및 100 μg/mL 스트렙토마이신이 포함된 DMEM 배지(이하, "Serum-free media" 또는 "SFM"이라 함)로 교체해 주었고, 아스코르브산 처리군의 경우 해당 배지에 30μg/mL의 아스코르브산을 첨가하였다.When both the ascorbic acid-treated and untreated groups reached a cell confluency of 80% or more, the cells were washed with phosphate-buffered saline (PBS, purchased from Thermo Scientific), and the culture medium was cultured at 100 units/mL. DMEM medium containing penicillin and 100 μg/mL streptomycin (hereinafter referred to as “serum-free media” or “SFM”) was replaced, and in the case of the ascorbic acid treatment group, 30 μg/mL ascorbic acid was added to the medium. did.
그리고 나서, 24시간 내지 72시간 배양하고 그 상층액(이하, 배양액)을 수거하였고, 수거가 완료된 세포는 세포 계수기를 이용해 세포수를 측정하였다.Then, the cells were cultured for 24 hours to 72 hours, and the supernatant (hereinafter, culture solution) was collected, and the cell count was measured using a cell counter for the collected cells.
한편, 마우스 대식세포주인 RAW 264.7은 한국세포주은행에서 구입하여 배양하였다. 세포 배양을 위해 10% 우태아 혈청 (fetal bovine serum: Thermo Scientific에서 구입) 및 1% 항생제-항진균제 (antibiotics-antimycotics: Thermo Scientific에서 구입)가 함유된 DMEM (Thermo Scientific에서 구입) 배지에 5% CO2, 37℃ 조건에서 계대 배양하였다. 또한, 인체 피부 섬유아세포(human dermal fibroblast) 및 이를 배양하기 위한 배지는 주식회사 세포바이오(대한민국 서울특별시 소재)에서 구입하여 5% CO2, 37℃조건에서 계대 배양하였다.Meanwhile, RAW 264.7, a mouse macrophage cell line, was purchased from the Korea Cell Line Bank and cultured. 5% CO in DMEM (purchased from Thermo Scientific) medium containing 10% fetal bovine serum (purchased from Thermo Scientific) and 1% antibiotics-antimycotics (purchased from Thermo Scientific) for cell culture 2 , subcultured at 37 ℃ condition. In addition, human dermal fibroblasts and a medium for culturing them were purchased from Cell Bio Co., Ltd. (Seoul, Korea) and subcultured at 5% CO 2 , 37°C conditions.
실시예 2: 엑소좀의 분리 및 정제Example 2: Isolation and purification of exosomes
실시예 1에서 회수한 배양액을 0.22 μm 필터로 여과하여 세포 잔해물, 노폐물 및 거대 입자 등의 불순물을 제거하였다. 여과된 배양액은 접선흐름여과법(Tangential Flow Filtration; TFF)을 이용한 분리 과정을 통해 엑소좀을 분리하였다. TFF 방법을 위한 필터로는 카트리지 필터(cartridge filter, 일명 hollow fiber filter; GE Healthcare 또는 Spectrum Labs에서 구입) 또는 카세트 필터(cassette filter; Pall 또는 Sartorius 또는 Merck Millipore에서 구입)를 사용하였다. TFF 필터는 다양한 분자량 차단(molecular weight cutoff; MWCO)에 의해 선택될 수 있다. 선택된 MWCO에 의해 선별적으로 엑소좀을 분리, 농축하였고, MWCO보다 작은 입자나 단백질, 지질, 핵산, 저분자 화합물 등은 제거하였다.The culture solution recovered in Example 1 was filtered with a 0.22 μm filter to remove impurities such as cell debris, wastes, and large particles. The filtered culture solution was separated from the exosomes through a separation process using tangential flow filtration (TFF). As a filter for the TFF method, a cartridge filter (aka hollow fiber filter; purchased from GE Healthcare or Spectrum Labs) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore) was used. TFF filters can be selected by various molecular weight cutoff (MWCO). Exosomes were selectively separated and concentrated by the selected MWCO, and particles smaller than the MWCO, proteins, lipids, nucleic acids, and low molecular weight compounds were removed.
엑소좀을 분리, 농축하기 위하여 MWCO 100,000 Da(Dalton), 300,000 Da, 500,000 Da 또는 750,000 Da의 TFF 필터, 또는 0.05 μm 크기의 TFF 필터를 사용하였다. 배양액을 TFF 방법을 이용하여 1/100 내지 1/10 정도의 부피가 될 때까지 농축하면서, MWCO보다 작은 물질들은 제거하여 엑소좀을 분리하였다.In order to isolate and concentrate the exosomes, a TFF filter of MWCO 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da, or a TFF filter having a size of 0.05 μm was used. While the culture medium was concentrated to a volume of about 1/100 to 1/10 using the TFF method, substances smaller than MWCO were removed to separate the exosomes.
분리, 농축된 엑소좀 용액은 TFF 방법을 이용하여 추가로 탈염과 버퍼교환 (diafiltration)을 수행하였다. 이때, 탈염과 버퍼교환은 연속적으로 수행(continuous diafiltration)하거나 단속적으로 수행(discontinuous diafiltration)하였으며, 시작 부피(starting volume)에 대하여 적어도 4배, 바람직하게는 6배 내지는 10배 이상, 보다 바람직하게는 12배 이상의 부피를 갖는 완충용액을 이용하여 수행하였다.The separated and concentrated exosome solution was further desalted and buffer exchanged (diafiltration) using the TFF method. At this time, desalting and buffer exchange were performed continuously (continuous diafiltration) or intermittently (discontinuous diafiltration), and at least 4 times, preferably 6 times to 10 times or more, more preferably with respect to the starting volume. This was performed using a buffer solution having a volume of 12 times or more.
한편, 줄기세포를 포함한 동물 유래 세포 배양액으로부터 엑소좀을 분리하는 방법으로는 전술한 바와 같은 분리방법 외에도 당업계에 알려진 다양한 방법을 이용할 수 있다. 예를 들어, 엑소좀의 분리를 위해, 초원심분리법 (ultracentrifugation), 초미세여과법(ultrafiltration), 밀도구배원심법(density gradient centrifugation), 크기 배제 크로마토그래피(size exclusion chromatography), 이온교환 크로마토그래피(ion exchange chromatography), 면역친화성 분리법(immunoaffinity capture), 미세유체기술 분리법(microfluidics-based isolation), 침전법(exosome precipitation), 총엑소좀 추출 키트(total exosome isolation kit), 또는 폴리머 기반 침전법(polymer based precipitation) 등 공지의 분리방법을 사용할 수 있다. 그러나 엑소좀의 분리방법은 전술한 바와 같은 방법들에 제한되는 것이 아니며, 당업계에서 사용되고 있거나 향후 사용될 수 있는 다양한 분리 방법이 채택가능한 것임은 물론이다.On the other hand, as a method of isolating the exosomes from an animal-derived cell culture medium including stem cells, various methods known in the art may be used in addition to the separation method as described above. For example, for the isolation of exosomes, ultracentrifugation, ultrafiltration, density gradient centrifugation, size exclusion chromatography, ion exchange chromatography ( ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, or polymer-based precipitation ( A known separation method such as polymer based precipitation) may be used. However, the separation method of the exosomes is not limited to the methods described above, and of course, various separation methods that are used in the art or that can be used in the future are acceptable.
실시예 3: 분리된 엑소좀의 특성 분석 및 생산성 평가Example 3: Characterization of isolated exosomes and evaluation of productivity
실시예 2에서 분리된 엑소좀은 나노입자 트랙킹 분석(nanoparticle tracking analysis: NTA; Malvern에서 구입)에 의해 입자의 크기와 농도를 측정하였다. 본 발명의 일 구체예의 분리방법에 따라 분리된 엑소좀의 NTA 분석 결과는 도 1A 및 도 1B에 도시하였다. 도 1A 및 도 1B에 도시된 바와 같이, 본 발명의 일 구체예에 따라 아스코르브산을 포함하는 배지에서 줄기세포를 전처리 및 배양한 후 수득된 엑소좀(이하, "아스코르브산 처리 엑소좀"이라 함; 도 1A)이 아스코르브산을 포함하지 않는 배지에서 줄기세포를 배양한 후 수득된 엑소좀(이하, "무처리 대조군 엑소좀"이라 함; 도 1B)에 비해 입자 크기 분포가 균일한 것을 알 수 있었다.The exosomes isolated in Example 2 were measured for particle size and concentration by nanoparticle tracking analysis ( NTA; purchased from Malvern). The NTA analysis results of the exosomes isolated according to the separation method of one embodiment of the present invention are shown in FIGS. 1A and 1B. 1A and 1B, the exosomes obtained after pretreatment and culturing of stem cells in a medium containing ascorbic acid according to an embodiment of the present invention (hereinafter, referred to as "ascorbic acid-treated exosomes") 1A) It can be seen that the particle size distribution is uniform compared to the exosomes obtained after culturing the stem cells in a medium not containing ascorbic acid (hereinafter, referred to as “untreated control exosomes”; FIG. 1B). there was.
따라서, 본 발명은 아스코르브산을 포함하는 배지에서 줄기세포를 전처리 및 배양함으로써 입자크기 분포가 균일한 엑소좀을 생산할 수 있다. Therefore, the present invention can produce exosomes having a uniform particle size distribution by pre-treating and culturing stem cells in a medium containing ascorbic acid.
실시예 4: 엑소좀의 항염 효능 평가Example 4: Evaluation of anti-inflammatory efficacy of exosomes
RAW 264.7 세포를 10% FBS를 포함한 DMEM 배지에 현탁시키고 이를 멀티웰 플레이트(multiwell plate)의 각 웰에 80 내지 90%의 밀집도(confluency)를 갖도록 분주하였다. 다음 날 LPS가 포함된 새로운 배지에 희석한 엑소좀(아스코르브산 처리 엑소좀 및 무처리 대조군 엑소좀 각각)을 적정 농도로 24시간 동안 처리하여 배양하였다. 이때, 아스코르브산에 의해 엑소좀의 생산성이 증가하는 것을 고려하여 아스코르브산 처리 엑소좀과 무처리 대조군 엑소좀을 동일 농도(1.5×109 입자/mL)로 조정하여 RAW 264.7 세포에 처리하였다. RAW 264.7 cells were suspended in DMEM medium containing 10% FBS and aliquoted to each well of a multiwell plate to have a confluency of 80 to 90%. The next day, exosomes (each of ascorbic acid-treated exosomes and untreated control exosomes) diluted in fresh medium containing LPS were treated and cultured at an appropriate concentration for 24 hours. At this time, in consideration of the increase in the productivity of exosomes by ascorbic acid, the ascorbic acid-treated exosomes and the untreated control exosomes were adjusted to the same concentration (1.5×10 9 particles/mL) and treated in RAW 264.7 cells.
배양이 끝난 후 배양 상층액(culture supernatant)을 취하고 배양 상층액 내에 존재하는 염증성 사이토카인을 측정하여 염증 반응을 확인하였다. 배양 상층액 내의 염증성 사이토카인 양은 IL-6 ELISA 키트를 이용하여 측정하였다. ELISA 키트 (R&D system에서 구입) 제조사의 매뉴얼에 따라, LPS만을 처리한 군에서의 IL-6 (염증성 사이토카인)의 생성량과, 아스코르브산 처리 엑소좀 및 무처리 대조군 엑소좀 각각과 함께 LPS가 처리된 군에서의 IL-6 (염증성 사이토카인)의 생성량을 확인하였다. After the culture was completed, the culture supernatant was taken and the inflammatory response was confirmed by measuring the inflammatory cytokines present in the culture supernatant. The amount of inflammatory cytokines in the culture supernatant was measured using an IL-6 ELISA kit. ELISA kit (purchased from R&D system) According to the manufacturer's manual, the production amount of IL-6 (inflammatory cytokine) in the group treated only with LPS, and LPS treatment together with ascorbic acid-treated exosomes and untreated control exosomes, respectively The production amount of IL-6 (inflammatory cytokine) in the treated group was confirmed.
도 2에 도시된 바와 같이, 생쥐의 대식세포인 RAW 264.7 세포에 LPS와 함께본 발명의 아스코르브산 처리 엑소좀을 처리한 경우 LPS와 함께 무처리 대조군 엑소좀을 처리한 경우에 비해 IL-6 (염증성 사이토카인)의 생성을 현저하게 억제시켰다. As shown in FIG. 2, when RAW 264.7 cells, which are macrophages of mice, were treated with the ascorbic acid-treated exosomes of the present invention together with LPS, compared to the case of treating the untreated control exosomes with LPS, IL-6 ( production of inflammatory cytokines) was significantly inhibited.
이러한 결과로부터, 본 발명에 따라 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 줄기세포를 전처리 및 배양하여 수득된 아스코르브산 처리 엑소좀은 항염, 창상 치료 또는 창상 치료 촉진 효능을 강화시키는 측면에서 무처리 대조군 엑소좀 보다 우수하다는 것을 알 수 있다. 따라서, 본 발명의 생리활성이 강화된 엑소좀의 생산방법 내지는 엑소좀의 생리활성 강화 방법은 임상이나 상업적으로 유용하게 활용될 수 있는 기술임을 알 수 있다. From these results, ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, an analog or derivative thereof according to the present invention are anti-inflammatory, wound treatment or wound treatment promoting efficacy It can be seen that it is superior to the treatment control exosomes. Therefore, it can be seen that the method of producing exosomes with enhanced physiological activity or the method of enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.
또한, 본 발명의 항염, 창상 치료 또는 창상 치료 촉진 효능이 강화된 아스코르브산 처리 엑소좀은 항염, 창상 치료 또는 창상 치료 촉진용 약학 조성물의 유효성분으로 유용하게 활용될 수 있다. In addition, the ascorbic acid-treated exosome with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention may be usefully utilized as an active ingredient of a pharmaceutical composition for anti-inflammatory, wound treatment or wound treatment promotion.
실시예 5: 엑소좀의 콜라겐 합성 증가 효능 평가Example 5: Evaluation of the effect of increasing collagen synthesis of exosomes
우태아 혈청이 포함된 DMEM 배지에 분산된 인체 피부 섬유아세포(주식회사 세포바이오에서 구입)를 멀티웰 플레이트에 분주한 후 24시간 배양하고 무혈청 배지에서 추가로 24시간 배양하였다. 이후, 아스코르브산 처리 엑소좀 및 무처리 대조군 엑소좀 각각을 농도별로 무혈청 배지에 희석한 후 인체 피부 섬유아세포에 처리하고 인체 피부 섬유아세포를 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다. 이때, 아스코르브산에 의해 엑소좀의 생산성이 증가하는 것을 고려하여 아스코르브산 처리 엑소좀과 무처리 대조군 엑소좀을 동일 농도로 조정하여 인체 피부 섬유아세포에 처리하였다. 저농도로 처리하는 경우, 아스코르브산 처리 엑소좀 및 무처리 대조군 엑소좀 각각을 5×108 입자(particles)/mL의 농도로 인체 피부 섬유아세포에 처리하였고, 고농도로 처리하는 경우, 아스코르브산 처리 엑소좀 및 무처리 대조군 엑소좀 각각을 1.5×109 입자(particles)/mL의 농도로 인체 피부 섬유아세포에 처리하였다.Human skin fibroblasts (purchased from Cell Bio Co., Ltd.) dispersed in DMEM medium containing fetal bovine serum were dispensed in a multi-well plate, cultured for 24 hours, and cultured for an additional 24 hours in serum-free medium. Thereafter, each of the ascorbic acid-treated exosomes and the untreated control exosomes were diluted in serum-free medium at different concentrations, treated with human skin fibroblasts, and cultured for 24 hours at 37° C., 5% CO 2 in an incubator. . At this time, in consideration of the increase in the productivity of exosomes by ascorbic acid, the ascorbic acid-treated exosomes and the untreated control exosomes were adjusted to the same concentration and treated with human skin fibroblasts. In the case of treatment with a low concentration, each of the ascorbic acid-treated exosome and the untreated control exosome was treated in human skin fibroblasts at a concentration of 5×10 8 particles/mL, and when treated at a high concentration, ascorbic acid-treated exo Each of the exosomes and untreated control exosomes was treated with human skin fibroblasts at a concentration of 1.5×10 9 particles/mL.
이후, 인체 피부 섬유아세포의 배양 상층액(culture supernatant)을 수거하여 원심분리한 후, 원심분리된 배양액을 준비하였다. 프로콜라겐 타입 IC-펩타이드(PIP)에 대한 EIA 키트(Takara에서 구입)를 이용하여 인체 피부 섬유아세포로부터 합성되어 배양액에 축적된 콜라겐 양을 측정하였다. 세포 파쇄액의 총 단백질 양은 BCA 단백질 정량 키트(ThermoFisher Scientific에서 구입)로 측정하였다. 측정된 콜라겐 양을 총 단백질 양으로 나누어 정규화하여 상대적인 콜라겐 양을 결정하였다. Thereafter, the culture supernatant of human skin fibroblasts was collected and centrifuged to prepare a centrifuged culture solution. The amount of collagen synthesized from human skin fibroblasts and accumulated in the culture medium was measured using an EIA kit for procollagen type IC-peptide (PIP) (purchased from Takara). The total protein amount in the cell lysate was measured with a BCA protein quantification kit (purchased from ThermoFisher Scientific). The measured collagen amount was divided by the total protein amount and normalized to determine the relative collagen amount.
도 3에 도시된 바와 같이, 24시간 배양 후 콜라겐 양을 측정한 결과, 본 발명의 아스코르브산 처리 엑소좀을 인체 피부 섬유아세포에 저농도 및 고농도로 처리한 경우 모두 무처리 대조군 엑소좀을 인체 피부 섬유아세포에 처리한 경우에 비해 콜라겐 합성을 현저히 증가시켰다. As shown in FIG. 3 , as a result of measuring the amount of collagen after culturing for 24 hours, when the ascorbic acid-treated exosomes of the present invention were treated at low and high concentrations in human skin fibroblasts, both the untreated control exosomes were treated with human skin fibers. Collagen synthesis was significantly increased compared to the case of treatment with the blast cells.
이러한 결과로부터, 본 발명에 따라 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 줄기세포를 전처리 및 배양하여 수득된 아스코르브산 처리 엑소좀은 콜라겐 합성 증가 효능 측면에서 무처리 대조군 엑소좀 보다 우수하다는 것을 알 수 있다. 따라서, 본 발명의 생리활성이 강화된 엑소좀의 생산방법 내지는 엑소좀의 생리활성 강화 방법은 임상이나 상업적으로 유용하게 활용될 수 있는 기술임을 알 수 있다. From these results, ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, an analog or derivative thereof according to the present invention are superior to the untreated control exosomes in terms of collagen synthesis increasing efficacy. Able to know. Therefore, it can be seen that the method of producing exosomes with enhanced physiological activity or the method of enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.
또한, 본 발명의 콜라겐 합성 증가 효능이 강화된 아스코르브산 처리 엑소좀은 무처리 대조군 엑소좀 보다 콜라겐 합성 증가와 관련된 생리활성 강화, 예를 들어 피부탄력개선, 주름개선 및/또는 피부재생 효능이 우수하고, 피부탄력개선, 주름개선 및/또는 피부재생용 화장료 조성물의 유효성분으로 유용하게 활용될 수 있다. In addition, the ascorbic acid-treated exosomes with enhanced collagen synthesis increasing efficacy of the present invention are superior to the untreated control exosomes in enhancing the physiological activity related to the increase in collagen synthesis, for example, improving skin elasticity, improving wrinkles and/or skin regeneration. And, it can be usefully used as an active ingredient in a cosmetic composition for skin elasticity improvement, wrinkle improvement and/or skin regeneration.
실시예 6: 엑소좀의 인체 피부 섬유아세포 증식 효능 평가Example 6: Evaluation of human skin fibroblast proliferation efficacy of exosomes
우태아 혈청이 포함된 DMEM 배지에 분산된 인체 피부 섬유아세포(주식회사 세포바이오에서 구입)를 멀티웰 플레이트에 분주한 후 24시간 배양하고 무혈청 배지에서 추가로 24시간 배양하였다. 이후, 아스코르브산 처리 엑소좀 및 무처리 대조군 엑소좀 각각과 Nuclight 시약(Sartorius에서 구입)을 무혈청 배지에 희석한 후 인체 피부 섬유아세포에 처리하고 인체 피부 섬유아세포를 37℃, 5% CO2 배양기에서 72시간 동안 배양하였다. 이때, 아스코르브산에 의해 엑소좀의 생산성이 증가하는 것을 고려하여 아스코르브산 처리 엑소좀과 무처리 대조군 엑소좀을 동일 농도(2×109 입자/mL)로 조정하여 인체 피부 섬유아세포를 처리하였다. 이후, Incucyte(Sartorius에서 구입)를 이용해 제조사의 매뉴얼에 따라 24시간 내지 72시간 동안의 세포증식능을 확인하였다. Human skin fibroblasts (purchased from Cell Bio Co., Ltd.) dispersed in DMEM medium containing fetal bovine serum were dispensed in a multi-well plate, cultured for 24 hours, and cultured for an additional 24 hours in serum-free medium. Thereafter, each of the ascorbic acid-treated exosomes and the untreated control exosomes and Nuclight reagent (purchased from Sartorius) were diluted in serum-free medium, treated with human skin fibroblasts, and human skin fibroblasts were incubated at 37°C, 5% CO 2 incubated for 72 hours. At this time, in consideration of the increase in the productivity of exosomes by ascorbic acid, human skin fibroblasts were treated by adjusting the ascorbic acid-treated exosomes and the untreated control exosomes to the same concentration (2×10 9 particles/mL). Then, using Incucyte (purchased from Sartorius) according to the manufacturer's manual, cell proliferation was confirmed for 24 hours to 72 hours.
도 4에 도시된 바와 같이, 72시간 배양 후 인체 피부 섬유아세포 증식능을 측정한 결과, 본 발명의 아스코르브산 처리 엑소좀을 인체 피부 섬유아세포에 처리한 경우 무처리 대조군 엑소좀을 인체 피부 섬유아세포에 처리한 경우에 비해 인체 피부 섬유아세포의 증식 효능을 현저히 증가시켰다. As shown in FIG. 4 , as a result of measuring the proliferative capacity of human skin fibroblasts after culturing for 72 hours, when the ascorbic acid-treated exosomes of the present invention were treated with human skin fibroblasts, the untreated control exosomes were applied to human skin fibroblasts. Compared to the treated case, the proliferative efficacy of human skin fibroblasts was significantly increased.
이러한 결과로부터, 본 발명에 따라 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 줄기세포를 전처리 및 배양하여 수득된 아스코르브산 처리 엑소좀은 인체 피부 섬유아세포의 증식 촉진 효능 측면에서 무처리 대조군 엑소좀 보다 우수하다는 것을 알 수 있다. 즉, 본 발명의 아스코르브산 처리 엑소좀은 무처리 대조군 엑소좀 보다 인체 피부 섬유아세포 증식 촉진과 관련된 생리활성 강화, 예를 들어 피부탄력개선, 주름개선, 피부재생, 창상 치료 및/또는 창상 치료 촉진 효능이 우수하다. 따라서, 본 발명의 생리활성이 강화된 엑소좀의 생산방법 내지는 엑소좀의 생리활성 강화 방법은 임상이나 상업적으로 유용하게 활용될 수 있는 기술임을 알 수 있다. From these results, ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, analogs or derivatives thereof according to the present invention are non-treated control exosomes in terms of the proliferation promoting efficacy of human skin fibroblasts. It can be seen that the better That is, the ascorbic acid-treated exosome of the present invention enhances physiological activity related to promotion of human skin fibroblast proliferation, for example, skin elasticity improvement, wrinkle improvement, skin regeneration, wound treatment and/or wound treatment promotion, than the untreated control exosome. Efficacy is excellent. Therefore, it can be seen that the method of producing exosomes with enhanced physiological activity or the method of enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.
또한, 본 발명의 아스코르브산 처리 엑소좀은 피부탄력개선, 주름개선 및/또는 피부재생용 화장료 조성물, 창상 치료 또는 창상 치료 촉진용 약학 조성물의 유효성분으로 유용하게 활용될 수 있다. In addition, the ascorbic acid-treated exosome of the present invention can be usefully used as an active ingredient in a cosmetic composition for skin elasticity improvement, wrinkle improvement and/or skin regeneration, and a pharmaceutical composition for wound treatment or wound treatment promotion.
이상, 본 발명을 상기 실시예를 들어 설명하였으나, 본 발명은 이에 제한되는 것이 아니다. 당업자라면 본 발명의 취지 및 범위를 벗어나지 않고 수정, 변경을 할 수 있으며 이러한 수정과 변경 또한 본 발명에 속하는 것임을 알 수 있을 것이다.In the above, the present invention has been described with reference to the above examples, but the present invention is not limited thereto. Those skilled in the art can make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.
Claims (23)
- 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하는 단계를 포함하는, 엑소좀의 생리활성 강화 방법.A method for enhancing the physiological activity of exosomes, comprising the step of culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof.
- 제1항에 있어서, According to claim 1,상기 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 무혈청 배지에서 추가로 배양하는 단계를 포함하는, 엑소좀의 생리활성 강화 방법.A method for enhancing the physiological activity of exosomes, comprising the step of further culturing the animal-derived cells in a serum-free medium containing ascorbic acid, an analog or a derivative thereof.
- 제1항 또는 제2항에 있어서, 3. The method of claim 1 or 2,상기 생리활성 강화는 항염, 창상 치료 또는 창상 치료 촉진 효능 강화인, 엑소좀의 생리활성 강화 방법.The enhancement of the physiological activity is anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancement, the method of enhancing the physiological activity of exosomes.
- 제1항 또는 제2항에 있어서,3. The method of claim 1 or 2,상기 생리활성 강화는 콜라겐 합성 증가 효능 강화인, 엑소좀의 생리활성 강화 방법. The enhancement of the physiological activity is a method of enhancing the physiological activity of exosomes, which is strengthening the effect of increasing collagen synthesis.
- 제1항 또는 제2항에 있어서,3. The method of claim 1 or 2,상기 아스코르브산, 이의 유사체 또는 유도체의 농도는 1 μg/mL 내지 300 μg/mL인, 엑소좀의 생리활성 강화 방법.The concentration of the ascorbic acid, its analogs or derivatives is 1 μg / mL to 300 μg / mL, the exosome physiological activity enhancement method.
- [규칙 제91조에 의한 정정 07.03.2022]
제5항에 있어서,상기 아스코르브산, 이의 유사체 또는 유도체의 농도는 10 μg/mL 내지 100 μg/mL인, 엑소좀의 생리활성 강화 방법.[Correction by Rule 91 07.03.2022]
6. The method of claim 5,The concentration of the ascorbic acid, its analogs or derivatives is 10 μg / mL to 100 μg / mL, the exosome physiological activity enhancement method. - 아스코르브산, 이의 유사체 또는 유도체를 포함하는, 동물세포 유래의 엑소좀의 생리활성 강화용 배지. A medium for enhancing the physiological activity of exosomes derived from animal cells, comprising ascorbic acid, an analog or derivative thereof.
- 제7항에 있어서, 8. The method of claim 7,상기 생리활성 강화는 항염, 창상 치료 또는 창상 치료 촉진 효능 강화인, 동물세포 유래의 엑소좀의 생리활성 강화용 배지.The enhancement of the physiological activity is anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancement, the medium for enhancing the physiological activity of exosomes derived from animal cells.
- 제7항에 있어서, 8. The method of claim 7,상기 생리활성 강화는 콜라겐 합성 증가 효능 강화인, 동물세포 유래의 엑소좀의 생리활성 강화용 배지.The enhancement of the physiological activity is a medium for enhancing the physiological activity of animal cell-derived exosomes, which is enhancement of the effect of increasing collagen synthesis.
- 제7항 내지 제9항 중 어느 한 항에 있어서, 10. The method according to any one of claims 7 to 9,상기 아스코르브산, 이의 유사체 또는 유도체의 농도는 1 μg/mL 내지 300 μg/mL인, 동물세포 유래의 엑소좀의 생리활성 강화용 배지. The concentration of the ascorbic acid, an analog or derivative thereof is 1 μg / mL to 300 μg / mL, a medium for enhancing the physiological activity of animal cells-derived exosomes.
- [규칙 제91조에 의한 정정 07.03.2022]
제10항에 있어서,상기 아스코르브산, 이의 유사체 또는 유도체의 농도는 10 μg/mL 내지 100 μg/mL인, 동물세포 유래의 엑소좀의 생리활성 강화용 배지.[Correction by Rule 91 07.03.2022]
11. The method of claim 10,The concentration of the ascorbic acid, an analog or derivative thereof is 10 μg / mL to 100 μg / mL, a medium for enhancing the physiological activity of animal cell-derived exosomes. - 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하여 수득된, 콜라겐 합성 증가 효능이 강화된 엑소좀을 포함하는 화장료 조성물.A cosmetic composition comprising exosomes with enhanced collagen synthesis efficacy obtained by culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof.
- 제12항에 있어서, 13. The method of claim 12,(a) 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하는 단계와, (b) 상기 동물 유래 세포로부터 분비 또는 방출된 엑소좀을 함유하는 배양액(conditioned media)을 수집한 후 수집한 배양액으로부터 엑소좀을 분리하는 단계를 수행하여 수득된, 콜라겐 합성 증가 효능이 강화된 엑소좀을 포함하는 화장료 조성물.(a) culturing the animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and (b) collecting a conditioned media containing exosomes secreted or released from the animal-derived cells. A cosmetic composition comprising exosomes having enhanced collagen synthesis increasing efficacy, obtained by performing the step of separating the exosomes from the collected culture medium.
- 제12항 또는 제13항에 있어서, 14. The method of claim 12 or 13,상기 아스코르브산, 이의 유사체 또는 유도체는 상기 배지 내에 1 μg/mL 내지 300 μg/mL의 농도로 포함되는, 화장료 조성물.The ascorbic acid, an analog or derivative thereof is contained in the medium at a concentration of 1 μg / mL to 300 μg / mL, cosmetic composition.
- [규칙 제91조에 의한 정정 07.03.2022]
제14항에 있어서,상기 아스코르브산, 이의 유사체 또는 유도체는 상기 배지 내에 10 μg/mL 내지 100 μg/mL의 농도로 포함되는, 화장료 조성물.[Correction by Rule 91 07.03.2022]
15. The method of claim 14,The ascorbic acid, its analog or derivative is contained in the medium at a concentration of 10 μg / mL to 100 μg / mL, the cosmetic composition. - 제12항 또는 제13항에 있어서, 14. The method of claim 12 or 13,피부탄력개선 효능, 주름개선 효능 또는 피부재생 효능 중 적어도 하나를 강화하는, 화장료 조성물.A cosmetic composition that enhances at least one of the skin elasticity improvement effect, the wrinkle improvement effect, or the skin regeneration effect.
- 제12항 또는 제13항에 있어서, 14. The method of claim 12 or 13,샴푸, 비누, 린스, 계면활성제-함유 클렌징, 크림, 로션, 연고, 토닉, 트리트먼트, 컨디셔너, 현탁액, 유탁액, 페이스트, 젤, 오일, 왁스, 스프레이, 에어졸, 미스트, 또는 파우더인, 화장료 조성물.Shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, mist, or powder cosmetic composition .
- 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하여 수득된, 항염, 창상 치료 또는 창상 치료 촉진 효능이 강화된 엑소좀을 포함하는, 항염, 창상 치료 또는 창상 치료 촉진용 약학 조성물.A pharmaceutical composition for promoting anti-inflammatory, wound treatment or wound treatment, comprising exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy, obtained by culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof .
- 제18항에 있어서, 19. The method of claim 18,(a) 동물 유래 세포를 아스코르브산, 이의 유사체 또는 유도체를 포함하는 배지에서 배양하는 단계와, (b) 상기 동물 유래 세포로부터 분비 또는 방출된 엑소좀을 함유하는 배양액(conditioned media)을 수집한 후 수집한 배양액으로부터 엑소좀을 분리하는 단계를 수행하여 수득된, 항염, 창상 치료 또는 창상 치료 촉진 효능이 강화된 엑소좀을 포함하는, 항염, 창상 치료 또는 창상 치료 촉진용 약학 조성물.(a) culturing the animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and (b) collecting a conditioned media containing exosomes secreted or released from the animal-derived cells. A pharmaceutical composition for promoting anti-inflammatory, wound treatment or wound treatment, comprising exosomes having enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy, obtained by performing the step of separating the exosomes from the collected culture medium.
- 제18항 또는 제19항에 있어서, 20. The method of claim 18 or 19,상기 아스코르브산, 이의 유사체 또는 유도체는 상기 배지 내에 1 μg/mL 내지 300 μg/mL의 농도로 포함되는, 항염, 창상 치료 또는 창상 치료 촉진용 약학 조성물.The ascorbic acid, its analog or derivative is contained in the medium at a concentration of 1 μg / mL to 300 μg / mL, anti-inflammatory, wound treatment or a pharmaceutical composition for promoting wound treatment.
- [규칙 제91조에 의한 정정 07.03.2022]
제20항에 있어서,상기 아스코르브산, 이의 유사체 또는 유도체는 상기 배지 내에 10 μg/mL 내지 100 μg/mL의 농도로 포함되는, 항염, 창상 치료 또는 창상 치료 촉진용 약학 조성물.[Correction by Rule 91 07.03.2022]
21. The method of claim 20,The ascorbic acid, analog or derivative thereof is contained in the medium at a concentration of 10 μg / mL to 100 μg / mL, anti-inflammatory, wound treatment or a pharmaceutical composition for promoting wound treatment. - 제18항 또는 제19항에 있어서, 20. The method of claim 18 or 19,주사, 마이크로니들링, 이온토포레시스, 도포, 또는 이들의 조합에 의해 투여 또는 처리되는, 항염, 창상 치료 또는 창상 치료 촉진용 약학 조성물. A pharmaceutical composition for anti-inflammatory, wound healing or accelerating wound healing, which is administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof.
- 제18항 또는 제19항에 있어서, 20. The method of claim 18 or 19,주사제형, 주입제형, 분무제형, 액상제형 또는 패취제형인 것을 특징으로 하는, 항염, 창상 치료 또는 창상 치료 촉진용 약학 조성물.An injection, injection, spray, liquid or patch formulation, anti-inflammatory, wound treatment or a pharmaceutical composition for promoting wound healing.
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JP2020528085A (en) * | 2017-07-24 | 2020-09-17 | 株式会社エキソステムテックExostemtech Co.,Ltd. | A composition for the prevention or treatment of osteoporosis containing exosomes extracted from stem cells as an active ingredient. |
KR102159915B1 (en) * | 2019-09-18 | 2020-09-24 | 주식회사 엑소코바이오 | Method for enhancing generation of exosomes and/or extracellular vesicles |
KR20200113225A (en) * | 2018-01-23 | 2020-10-06 | 노바셀라 가부시키가이샤 | Composition for treating skin diseases |
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US20180147420A1 (en) * | 2015-07-31 | 2018-05-31 | Exoceuticals, Inc. | Exosome compositions and methods for preparation and use thereof for regulating and conditioning skin and hair |
KR20180098475A (en) * | 2017-02-24 | 2018-09-04 | (주)인핸스드바이오 | Nanovesicle derived from stem cell and composition of promoting hair growth and restoration comprising the same |
JP2020528085A (en) * | 2017-07-24 | 2020-09-17 | 株式会社エキソステムテックExostemtech Co.,Ltd. | A composition for the prevention or treatment of osteoporosis containing exosomes extracted from stem cells as an active ingredient. |
KR20200113225A (en) * | 2018-01-23 | 2020-10-06 | 노바셀라 가부시키가이샤 | Composition for treating skin diseases |
KR102159915B1 (en) * | 2019-09-18 | 2020-09-24 | 주식회사 엑소코바이오 | Method for enhancing generation of exosomes and/or extracellular vesicles |
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